Anti-tau antibodies and methods of use

ABSTRACT

The invention provides anti-Tau antibodies and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/420,673, filed May 23, 2019, which is a continuation of InternationalApplication No. PCT/US2017/064869, filed Dec. 6, 2017, which claims thebenefit of priority of U.S. Provisional Application No. 62/431,180,filed Dec. 7, 2016, each of which is incorporated by reference herein inits entirety for any purpose.

FIELD OF THE INVENTION

The present invention relates to anti-Tau antibodies and methods ofusing the same.

BACKGROUND

Neurofibrillary tangles and neuropil threads (NTs) are the majorneuropathological hallmarks of Alzheimer's Disease (AD). NTs arecomposed of the microtubule-associated Tau protein that has undergonepost-translational modifications including phosphorylation, and developby aggregation of hyperphosphorylated Tau conformers. AD shares thispathology with many neurodegenerative tauopathies, in particularly withcertain types of frontotemporal dementia (FTD). Tau protein appears tobe a major player in the cognitive demise in AD and relatedneurodegenerative tauopathies.

Therapeutic approaches that target Tau protein are scarce and comprisemainly inhibitors of the kinases that are thought to increase thephosphorylation of Tau to pathological levels, and compounds that blockthe cytoplasmic aggregation of hyper-phosphorylated Tau protein. Theseapproaches suffer various draw-backs of specificity and efficacy. Thereis a need for additional therapeutic agents that target the pathologicalprotein conformers that are known or presumed to cause neurodegenerativedisorders.

SUMMARY

The invention provides anti-Tau antibodies and methods of using thesame. In some embodiments, antibodies with high affinity for human andcynomolgus monkey Tau are provided. In some embodiments, the antibodieshave an affinity for human Tau of less than 1 nM or less than 0.5 nM orless than 0.3 nM as measured, for example, by surface plasmon resonance.

In some embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody binds to monomeric Tau, oligomeric Tau,non-phosphorylated Tau, and phosphorylated Tau. In some embodiments, theantibody binds an epitope within amino acids 2 to 24 of mature humanTau. In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a human, humanized, or chimeric antibody.In some embodiments, the antibody is an antibody fragment that bindshuman Tau. In some embodiments, the human Tau comprises the sequence ofSEQ ID NO: 2.

In some embodiments, the antibody comprises HVR-H1 comprising an aminoacid sequence of SEQ ID NO: 605; HVR-H2 comprising an amino acidsequence of SEQ ID NO: 606; and HVR-H3 comprising an amino acid sequenceof SEQ ID NO: 607.

In some embodiments, the antibody comprises HVR-L1 comprising an aminoacid sequence of SEQ ID NO: 608; HVR-L2 comprising an amino acidsequence of SEQ ID NO: 609; and HVR-L3 comprising an amino acid sequenceof SEQ ID NO: 610.

In some embodiments, the antibody comprises HVR-H1 comprising an aminoacid sequence of SEQ ID NO: 605; HVR-H2 comprising an amino acidsequence of SEQ ID NO: 606; HVR-H3 comprising an amino acid sequence ofSEQ ID NO: 607; HVR-L1 comprising an amino acid sequence of SEQ ID NO:608; HVR-L2 comprising an amino acid sequence of SEQ ID NO: 609; andHVR-L3 comprising an amino acid sequence of SEQ ID NO: 610.

In some embodiments, the antibody comprises:

-   -   a) a heavy chain variable region (VH) comprising a sequence that        is at least 95% identical to SEQ ID NO: 603;    -   b) a light chain variable region (VL) comprising a sequence that        is at least 95% identical to SEQ ID NO: 604;    -   c) a VH as in (a) and a VL as in (b);    -   d) a heavy chain variable region (VH) comprising a sequence that        is at least 95% identical to SEQ ID NO: 614;    -   e) a light chain variable region (VL) comprising a sequence that        is at least 95% identical to SEQ ID NO: 615;    -   f) a VH as in (d) and a VL as in (e);    -   g) a heavy chain variable region (VH) comprising a sequence that        is at least 95% identical to SEQ ID NO: 619;    -   h) a light chain variable region (VL) comprising a sequence that        is at least 95% identical to SEQ ID NO: 620;    -   i) a VH as in (g) and a VL as in (h).

In some embodiments, the antibody comprises:

-   -   a) a heavy chain variable region (VH) comprising SEQ ID NO: 603;    -   b) a light chain variable region (VL) comprising SEQ ID NO: 604;    -   c) a VH as in (a) and a VL as in (b);    -   d) a heavy chain variable region (VH) comprising the sequence of        SEQ ID NO: 614;    -   e) a light chain variable region (VL) comprising the sequence of        SEQ ID NO: 615;    -   f) a VH as in (d) and a VL as in (e);    -   g) a heavy chain variable region (VH) comprising the sequence of        SEQ ID NO: 619;    -   h) a light chain variable region (VL) comprising the sequence of        SEQ ID NO: 620;    -   i) a VH as in (g) and a VL as in (h).

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence selected from SEQ ID NOs: 603, 614, and619; and a light chain variable region comprising a sequence selectedfrom SEQ ID NOs: 604, 615, and 620. In some embodiments, the antibodycomprises a heavy chain variable region comprising a sequence selectedfrom SEQ ID NOs: 340, 603, 614, and 619; and a light chain variableregion comprising a sequence selected from SEQ ID NOs: 604, 615, and620. In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence selected from SEQ ID NOs: 603, 614, and619; and a light chain variable region comprising a sequence selectedfrom SEQ ID NOs: 341, 604, 615, and 620.

In some embodiments, the antibody comprises (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 603 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:604; (b) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 614 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 615; or (c) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 619 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:620.

In some embodiments, the antibody comprises (a) a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a lightchain comprising the amino acid sequence of SEQ ID NO: 613; (b) a heavychain comprising the amino acid sequence of SEQ ID NO: 616 or SEQ ID NO:617 and a light chain comprising the amino acid sequence of SEQ ID NO:618; or (c) a heavy chain comprising the amino acid sequence of SEQ IDNO: 621 or SEQ ID NO: 622 and a light chain comprising the amino acidsequence of SEQ ID NO: 623.

In some embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a lightchain comprising the amino acid sequence of SEQ ID NO: 613. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 611 and a light chain comprising the amino acidsequence of SEQ ID NO: 613. In some embodiments, an isolated antibodythat binds to human Tau is provided, wherein the antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 612 and alight chain comprising the amino acid sequence of SEQ ID NO: 613. Insome embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a lightchain consisting of the amino acid sequence of SEQ ID NO: 613. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain consisting of the aminoacid sequence of SEQ ID NO: 611 and a light chain consisting of theamino acid sequence of SEQ ID NO: 613. In some embodiments, an isolatedantibody that binds to human Tau is provided, wherein the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 612 and a light chain consisting of the amino acid sequence of SEQID NO: 613.

In some embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 616 or 617 and a light chaincomprising the amino acid sequence of SEQ ID NO: 618. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 616 and a light chain comprising the amino acidsequence of SEQ ID NO: 618. In some embodiments, an isolated antibodythat binds to human Tau is provided, wherein the antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 617 and alight chain comprising the amino acid sequence of SEQ ID NO: 618. Insome embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 616 or SEQ ID NO: 617 and a lightchain consisting of the amino acid sequence of SEQ ID NO: 618. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain consisting of the aminoacid sequence of SEQ ID NO: 616 and a light chain consisting of theamino acid sequence of SEQ ID NO: 618. In some embodiments, an isolatedantibody that binds to human Tau is provided, wherein the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 617 and a light chain consisting of the amino acid sequence of SEQID NO: 618.

In some embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a lightchain comprising the amino acid sequence of SEQ ID NO: 623. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 621 and a light chain comprising the amino acidsequence of SEQ ID NO: 623. In some embodiments, an isolated antibodythat binds to human Tau is provided, wherein the antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 622 and alight chain comprising the amino acid sequence of SEQ ID NO: 623. Insome embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a lightchain consisting of the amino acid sequence of SEQ ID NO: 623. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain consisting of the aminoacid sequence of SEQ ID NO: 621 and a light chain consisting of theamino acid sequence of SEQ ID NO: 623. In some embodiments, an isolatedantibody that binds to human Tau is provided, wherein the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 622 and a light chain consisting of the amino acid sequence of SEQID NO: 623.

In any of the embodiments described herein, the antibody may be an IgG1or an IgG4 antibody. In any of the embodiments described herein, theantibody may be an IgG4 antibody. In some such embodiments, the antibodycomprises M252Y, S254T, and T256E mutations. In any of the embodimentsdescribed herein, the antibody may comprise an S228P mutation. In any ofthe embodiments described herein, the antibody may comprise S228P,M252Y, S254T, and T256E mutations. In any of the embodiments describedherein, the antibody may be an IgG4 antibody comprising S228P, M252Y,S254T, and T256E mutations. In some embodiments, the antibody is anantibody fragment. In any of the embodiments described herein, theantibody may be an IgG4 antibody comprising S228P, M252Y, S254T, andT256E mutations, and lacking the C-terminal lysine (des-K) of the heavychain constant region. The C-terminal lysine of the heavy chain constantregion may be removed, for example, during purification of the antibodyor by recombinant engineering of the nucleic acid encoding the antibodysuch that the C-terminal lysine is not encoded.

In some embodiments, an isolated antibody that binds human Tau isprovided, wherein the antibody binds each of monomeric Tau,phosphorylated Tau, non-phosphorylated Tau, and oligomeric Tau with aK_(D) of less than 100 nM, less than 75 nM, less than 50 nM, less than10 nM, less than 5 nM, or less than 1 nM. In some embodiments, theantibody binds cynomolgus monkey Tau. In some embodiments, the antibodybinds to human monomeric Tau and/or cynomolgus monkey monomeric Tau witha K_(D) of less than 1 nM.

In some embodiments, an isolated nucleic acid is provided, wherein theisolated nucleic acid encodes an antibody described herein. In someembodiments, a host cell is provided, wherein the host cell comprises anisolated nucleic acid that encodes an antibody described herein. In someembodiments, a method of producing an antibody is provided, comprisingculturing the host cell under conditions suitable for producing theantibody.

In some embodiments, an immunoconjugate is provided, wherein theimmunoconjugate comprises an isolated antibody described herein and atherapeutic agent. In some embodiments, a labeled antibody is provided,comprising an antibody described herein and a detectable label.

In some embodiments, a pharmaceutical composition is provided,comprising an isolated antibody described herein and a pharmaceuticallyacceptable carrier.

In some embodiments, a method of treating a Tau protein associateddisease is provided, comprising administering to an individual with aTau protein related disease an antibody described herein or apharmaceutical composition comprising an antibody described herein. Insome embodiments, the Tau protein associated disease is a tauopathy. Insome embodiments, the tauopathy is a neurodegenerative tauopathy. Insome embodiments, the tauopathy is selected from Alzheimer's Disease,amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jacobdisease, Dementia pugilistica, Down's Syndrome,Gerstmann-Straussler-Scheinker disease, inclusion-body myositis, prionprotein cerebral amyloid angiopathy, traumatic brain injury, amyotrophiclateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanianmotor neuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangleswith calcification, frontotetemporal dementia, frontotemporal dementiawith parkinsonism linked to chromosome 17, Hallevorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy. In some embodiments, the tauopathy isAlzheimer's disease or progressive supranuclear palsy. In someembodiments, the Tau protein associated disease is selected from PART(primary age-related Tauopathy), tangle predominant dementia, subacutesclerosis panencephalopathy, chronic traumatic encephalopathy (CTE),white matter tauopathy with globular glial inclusions, Lewy bodydementia (LBD), mild cognitive impairment (MCI), glaucoma, familialBritish dementia, familiar Danish dementia, Guadeloupean Parkinsonism,neurodegeneration with brain iron accumulation, SLC9A6-related mentalretardation, multiple sclerosis, HIV-related dementia, senile cardiacamyloidosis, and Huntington's disease.

In some embodiments, a method of retaining or increasing cognitivememory capacity or slowing memory loss in an individual is provided,comprising administering an antibody described herein or apharmaceutical composition comprising an antibody described herein.

In some embodiments, a method of reducing the level of Tau protein,non-phosphorylated Tau protein, phosphorylated Tau protein, orhyperphosphorylated Tau protein in an individual is provided, comprisingadministering an antibody described herein or a pharmaceuticalcomposition comprising an antibody described herein.

In some embodiments, an isolated antibody described herein is providedfor use as a medicament. In some embodiments, an isolated antibodydescribed herein is provided for use in treating a tauopathy in anindividual. In some embodiments, the tauopathy is a neurodegenerativetauopathy. In some embodiments, the tauopathy is selected fromAlzheimer's Disease, amyotrophic lateral sclerosis, Parkinson's disease,Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome,Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis, prionprotein cerebral amyloid angiopathy, traumatic brain injury, amyotrophiclateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanianmotor neuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangleswith calcification, frontotetemporal dementia, frontotemporal dementiawith parkinsonism linked to chromosome 17, Hallevorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy. In some embodiments, the tauopathy isAlzheimer's disease or progressive supranuclear palsy. In someembodiments, the Tau protein associated disease is selected from PART(primary age-related Tauopathy), tangle predominant dementia, subacutesclerosis panencephalopathy, chronic traumatic encephalopathy (CTE),white matter tauopathy with globular glial inclusions, Lewy bodydementia (LBD), mild cognitive impairment (MCI), glaucoma, familialBritish dementia, familiar Danish dementia, Guadeloupean Parkinsonism,neurodegeneration with brain iron accumulation, SLC9A6-related mentalretardation, multiple sclerosis, HIV-related dementia, senile cardiacamyloidosis, and Huntington's disease.

In some embodiments, an isolated antibody described herein is providedfor use in retaining or increasing cognitive memory capacity or slowingmemory loss in an individual. In some embodiments, an isolated antibodydescribed herein is provided for use in reducing the level of Tauprotein, phosphorylated Tau protein, non-phosphorylated Tau protein, orhyperphosphorylated Tau protein in an individual.

In some embodiments, use of an antibody described herein is provided formanufacture of a medicament for treating a Tau protein associateddisease in an individual. In some embodiments, the Tau protein associatedisease is a tauopathy. In some embodiments, the tauopathy is aneurodegenerative tauopathy. In some embodiments, the tauopathy isselected from Alzheimer's Disease, amyotrophic lateral sclerosis,Parkinson's disease, Creutzfeldt-Jacob disease, Dementia pugilistica,Down's Syndrome, Gerstmann-Sträussler-Scheinker disease, inclusion-bodymyositis, prion protein cerebral amyloid angiopathy, traumatic braininjury, amyotrophic lateral sclerosis/parkinsonism-dementia complex ofGuam, Non-Guamanian motor neuron disease with neurofibrillary tangles,argyrophilic grain dementia, corticobasal degeneration, diffuseneurofibrillary tangles with calcification, frontotetemporal dementia,frontotemporal dementia with parkinsonism linked to chromosome 17,Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick diseasetype C, Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy. In some embodiments, the tauopathy isAlzheimer's disease or progressive supranuclear palsy. In someembodiments, the Tau protein associated disease is selected from PART(primary age-related Tauopathy), tangle predominant dementia, subacutesclerosis panencephalopathy, chronic traumatic encephalopathy (CTE),white matter tauopathy with globular glial inclusions, Lewy bodydementia (LBD), mild cognitive impairment (MCI), glaucoma, familialBritish dementia, familiar Danish dementia, Guadeloupean Parkinsonism,neurodegeneration with brain iron accumulation, SLC9A6-related mentalretardation, multiple sclerosis, HIV-related dementia, senile cardiacamyloidosis, and Huntington's disease.

In some embodiments, use of an antibody described herein is provided formanufacture of a medicament for retaining or increasing cognitive memorycapacity or slowing memory loss in an individual.

In some embodiments, a method of detecting neurofibrillary tangles,neuropil threads, or dystrophic neuritis is provided, comprisingcontacting a sample with an antibody described herein. In someembodiments, the sample is a brain sample, a cerebrospinal fluid sample,or a blood sample.

In any of the embodiments described herein, a method or use may compriseadministering an antibody described herein in combination with at leastone additional therapy. Non-limiting examples of additional therapiesinclude neurological drugs, corticosteroids, antibiotics, antiviralagents, and other therapeutic agents. Such other therapeutic agentsinclude, but are not limited to, other anti-Tau antibodies, antibodiesagainst amyloid-beta, antibodies against beta-secretase 1 (“BACE1”), andinhibitors of beta-secretase 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-F. Binding of antibodies to hyperphosphorylated Tau (pTau) wascompared to non-phosphorylated Tau using an ELISA. Results are expressedin optical densities (O.D.).

FIG. 2A-E. Binding of antibodies to oligomeric Tau was assessed using anoligo- and monoTau capture ELISA. Results are expressed in opticaldensities (O.D.).

FIG. 3. The three panTau antibodies tested show binding to soluble Tauin brain lysates from Alzheimer's disease (AD) and matched controldonors using a Western blot (WB) assay. Protein extracts from AD andcontrol brain lysates, and six isoforms of recombinant human Tau, wererun on SDS-PAGE and membranes blotted with three panTau antibodies(37D3-H9, 94B2-C1, and 125B11-H3). Lanes with AD samples are labeled asAD18, AD24, and AD27, and lanes with control samples are labeled as C25and C21. The lanes run with six isoforms of recombinant human Tau arelabeled as hTau ladder.

FIG. 4A-C. PanTau antibodies show binding to soluble Tau in brainlysates from AD and matched control donors using a Tau capture ELISA.Data is shown for three panTau antibodies, 37D3-H9, 94B2-C1, and125B11-H3. Results are expressed in optical densities (O.D.), with meanvalues±SD, N=2.

FIG. 5. Sensorgrams showing 37D3-H9 binding as a Fab (left panel) and asan IgG (right panel) to human Tau monomer covalently coupled to aBiacore chip surface. A 1:1 binding model has been applied and is shownas an overlay. The x-axis indicates time (units=seconds). The y-axisindicated Resonance Units (RU).

FIG. 6. Overlaid sensorgrams showing binding of hu37D3-H9.v5 samples t=0(left panel) and t=2 weeks (right panel) to human Tau monomer at 3.1,6.3, 12.5, 25, 25, 50 and 100 nM. A 1:1 binding model has been appliedand is also shown in this figure. The x-axis indicates time(units=seconds). The y-axis indicated Resonance Units (RU).

FIG. 7. Binding of hu37D3-H9.v5 and hu37D3-H9.v5 N28D to monomeric Tauindividually (left panel shows hu37D3-H9.v5 and middle panel showshu37D3-H9.v5 N28D) and mixed at a 1:1 ratio (right panel). The x-axisindicates time (units=seconds). The y-axis indicated Resonance Units(RU).

FIG. 8A-D. Affinity, stability index and sequences of the ninety 37D3-H9variants screened for potential improved stability. For clarity, valuesobtained with an unstressed control antibody (hu37D3-H9.v5 hIgG1) run atthe beginning, middle and end of each experiment are shown in bothsections of the table.

FIG. 9. Structural model of the 37D3-H9 Fv region showing the positionsof residues 28 to 33 (NGNTYF motif) of the light chain and relativepositions of residues 28 and 33. Note that residue 33, mutated inhu37D3.v28.A4 to Leu, is not nearby the unstable Asn-28 residue. Thedotted line shows a hydrogen bond between residues Asn-28 and Tyr-32.Figure generated using MOE software package (Chemical Computing Group).

FIG. 10 shows pharmacokinetics of anti-Tau antibody 37D3-H9 in micefollowing a single 10 mg/kg intravenous or intraperitoneal injection.

FIG. 11 shows pharmacokinetics of hu37D3.v28.A4 hIgG4-S228P andhu37D3.v28.A4 hIgG4-S228P.YTE in cynomolgus monkeys following a singleIV bolus injection at a dose of 1 mg/kg.

FIG. 12A-C. Binding of certain anti-Tau antibodies to Tau fragments. (A)Binding of certain anti-Tau antibodies to Tau fragments 1-15, 10-24,19-33, 28-42, 37-51, and 46-60 is shown. (B) Binding of antibody 37D3-H9mIgG2a to Tau fragments 10-44, 10-24, 2-24, 2-34, and full-length Tau.(C) Binding of antibody hu37D3-H9.v5 hIgG1 to Tau fragments 10-44,10-24, 2-24, 2-34, and full-length Tau.

FIG. 13A-B. Effect of effector function on Tau toxicity inneuron-microglia co-cultures. (A) Percent MAP2 fragmentation inco-cultures contacted with various antibodies and oligomeric Tau. (B)Images of neurons (top panels) and neuron-microglia co-cultures (bottompanels) contacted with various antibodies and oligomeric Tau.

FIG. 14. pTau212/214 levels in the hippocampus of mice administeredanti-tau 37D3-H9 WT IgG2a or anti-tau 37D3-H9 DANG IgG2.

FIG. 15. Comparison of human and cynomolgus monkey Tau sequences. Theepitope for antibody 37D3-H9 is indicated.

FIG. 16 shows pharmacokinetics of anti-Tau antibody 94B2-C1 in micefollowing a single 10 mg/kg intravenous or intraperitoneal injection.

FIG. 17 shows pharmacokinetics of anti-Tau antibody 125B11-H3 in micefollowing a single 10 mg/kg intravenous or intraperitoneal injection.

FIG. 18 shows an alignment of the kappa 1 light chain variable regionsof hu37D3-H9.v1, hu37D3-H9.v39, hu37D3-H9.v40, and hu37D3-H9.v41.

FIG. 19A-B show plasma antibody concentration (A) and CSF antibodyconcentration (B) in cynomolgus monkeys following a single IV injectionof the indicated antibody at 50 mg/kg.

FIG. 20 shows plasma total Tau concentration and plasma antibodyconcentration in cynomolgus monkeys following a single IV injection ofthe indicated antibody at 50 mg/kg.

FIGS. 21A-D show antibody concentration in various regions of cynomolgusmonkey brain 2 days and 10 days following a single IV injection ofhu37D3.v28.A4 hIgG4-S228P (A) and hu37D3.v28.A4 hIgG4-S228P.YTE (B) at50 mg/kg; average antibody concentration in brain (C); % brain:plasmaantibody concentration (D).

FIGS. 22A-B show the concentration of antibody in cynomolgus monkeybrain at various time points following a single IV injection of theindicated antibody at 50 mg/kg, plotted in logarithmic (A) and linear(B) scale.

FIGS. 23A-E show the concentration of antibody in the hippocampus (A),cerebellum (B), frontal cortex (C), CSF (D), and plasma (E) ofcynomolgus monkeys at various time points following a single IVinjection of the indicated antibody at 50 mg/kg.

FIGS. 24A-B show average (A) and individual (B) plasma total Tauconcentration over time in cynomolgus monkeys following a single IVinjection of the indicated antibody at 50 mg/kg.

FIG. 25A-B shows sequence alignment between the affinity-maturedhu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93 antibodies versus theparent hu37D3-H9.v28.A4 antibody. Amino acid differences are shaded inblack.

FIGS. 26A-B show affinity measurements of the hu37D3-H9.v76 for humantau monomer (A) and cynomolgus monkey tau monomer (B).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)). Affinity can be measured by common methods known inthe art, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-Tau antibody” and “an antibody that binds to Tau” referto an antibody that is capable of binding Tau with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting Tau. In some embodiments, the extent of binding of ananti-Tau antibody to an unrelated, non-Tau protein is less than about10% of the binding of the antibody to Tau as measured, e.g., by aradioimmunoassay (MA). In certain embodiments, an antibody that binds toTau has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³M). In certain embodiments, ananti-Tau antibody binds to an epitope of Tau that is conserved among Taufrom different species. The term “anti-Tau antibody” and “antibody thatbinds to Tau,” as used herein, refers to an antibody that bindsmonomeric Tau, oligomeric Tau, and/or phosphorylated Tau, unlessspecifically indicated otherwise. In some such embodiments, the anti-Tauantibody binds to monomeric Tau, oligomeric Tau, non-phosphorylated Tau,and phosphorylated Tau with comparable affinities, such as withaffinities that differ by no more than 50-fold from one another. In someembodiments, an antibody that binds monomeric Tau, oligomeric Tau,non-phosphorylated Tau, and phosphorylated Tau is referred to as a“pan-Tau antibody.”

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In some embodiments, a human IgG heavy chain Fcregion extends from Cys226, or from Pro230, to the carboxyl-terminus ofthe heavy chain. However, the C-terminal lysine (Lys447) of the Fcregion may or may not be present. Unless otherwise specified herein,numbering of amino acid residues in the Fc region or constant region isaccording to the EU numbering system, also called the EU index, asdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. Insome embodiments, for the VL, the subgroup is subgroup kappa I as inKabat et al., supra. In some embodiments, for the VH, the subgroup issubgroup III as in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-Tau antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “Tau,” as used herein, refers to any native Tau protein fromany vertebrate source, including mammals such as primates (e.g., humans)and rodents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed Tau as well as any form of Tauthat results from processing in the cell. The term also encompassesnaturally occurring variants of Tau, e.g., splice variants or allelicvariants.

The term “pTau,” as used herein, refers to Tau in which a serine, athreonine or a tyrosine residue is phosphorylated by a protein kinase bythe addition of a covalently bound phosphate group. In some embodiments,pTau is phosphorylated on a serine or on a threonine residue. In someembodiments, pTau is phosphorylated on Serine at position 409 and/orSerine at position 404. In some embodiments, pTau is phosphorylated onSerine at position 409.

The terms “soluble Tau” or “soluble Tau protein,” as used herein, referto proteins consisting of both completely solubilized Tauprotein/peptide monomers or of Tau-like peptides/proteins, or ofmodified or truncated Tau peptides/proteins or of other derivates of Taupeptides/proteins monomers, and of Tau protein oligomers. “Soluble Tau”excludes particularly neurofibrillary tangles (NFT).

The term “insoluble Tau,” as used herein, refers to multiple aggregatedmonomers of Tau peptides or proteins, or of Tau-like peptides/proteins,or of modified or truncated Tau peptides/proteins or of other derivatesof Tau peptides/proteins forming oligomeric or polymeric structureswhich are insoluble both in vitro in aqueous medium and in vivo in themammalian or human body more particularly in the brain, but particularlyto multiple aggregated monomers of Tau or of modified or truncated Taupeptides/proteins or of derivatives thereof, which are insoluble in themammalian or human body more particularly in the brain, respectively.“Insoluble Tau” particularly includes neurofibrillary tangles (NFT).

The terms “monomeric Tau” or “Tau monomer,” as used herein, refer tocompletely solubilized Tau proteins without aggregated complexes inaqueous medium.

The terms “aggregated Tau”, “oligomeric Tau” and “Tau oligomer,” as usedherein, refer to multiple aggregated monomers of Tau peptides orproteins, or of Tau-like peptides/proteins, or of modified or truncatedTau peptides/proteins or of other derivates of Tau peptides/proteinsforming oligomeric or polymeric structures which are insoluble orsoluble both in vitro in aqueous medium and in vivo in the mammalian orhuman body more particularly in the brain, but particularly to multipleaggregated monomers of Tau or of modified or truncated Taupeptides/proteins or of derivatives thereof, which are insoluble orsoluble in the mammalian or human body more particularly in the brain,respectively.

The terms “pTau PHF”, “PHF”, and “paired helical filaments,” are usedherein synonymously, refer to pairs of filaments wound into helices witha periodicity of 160 nm visible on electron microscopy. Width variesbetween 10 and 22 nm. PHF are the predominant structures inneurofibrillary tangles of Alzheimer's Disease (AD) and neuropilthreads. PHF may also be seen in some but not all dystrophic neuritesassociated with neuritic plaques. The major component of PHF is ahyperphosphorylated form of microtubule-associated protein tau. PHF maybe partially composed of disulfide-linked antiparallelhyper-phosphorylated Tau proteins. PHF Tau may be truncated of itsC-terminal 20 amino acid residues. The mechanisms underlying PHFformation are uncertain but hyper-phosphorylation of Tau may disengageit from microtubules, increasing the soluble pool of Tau from which PHFcan be formed inside neurons.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “early Alzheimer's Disease” or “early AD” as used herein (e.g.,a “patient diagnosed with early AD” or a “patient suffering from earlyAD”) includes patients with mild cognitive impairement, such as a memorydeficit, due to AD and patients having AD biomarkers, for exampleamyloid positive patients.

The term “mild Alzheimer's Disease” or “mild AD” as used herein (e.g., a“patient diagnosed with mild AD”) refers to a stage of AD characterizedby an MMSE score of 20 to 26.

The term “mild to moderate Alzheimer's Disease” or “mild to moderate AD”as used herein encompasses both mild and moderate AD, and ischaracterized by an MMSE score of 18 to 26.

The term “moderate Alzheimer's Disease” or “moderate AD” as used herein(e.g., a “patient diagnosed with moderate AD”) refers to a stage of ADcharacterized by an MMSE score of 18 to 19.

The term “MMSE” refers to the Mini Mental State Examination, whichprovides a score between 1 and 30. See Folstein, et al., 1975, J.Psychiatr. Res. 12:189-98. Scores of 26 and lower are generallyconsidered to be indicative of a deficit. The lower the numerical scoreon the MMSE, the greater the tested patient's deficit or impairmentrelative to another individual with a lower score. An increase in MMSEscore may be indicative of improvement in the patient's condition,whereas a decrease in MMSE score may denote worsening in the patient'scondition.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

Antibodies that bind Tau are provided. In some embodiments, an antibodyprovided herein binds human monomeric Tau with a K_(D) of less than 1nM, or less than 0.5 nM. In some embodiments, an antibody providedherein binds cynomolgus monkey Tau with a K_(D) of less than 1 nM, orless than 0.5 nM. In some embodiments, K_(D) is determined by surfaceplasmon resonance at 37° C. In some embodiments, an antibody of theinvention binds Tau binds monomeric Tau, oligomeric Tau,non-phosphorylated Tau, and phosphorylated Tau. In some embodiments, anantibody of the invention binds to an epitope within amino acids 2 to 24of mature human Tau. In some embodiments, an antibody of the inventionbinds to an epitope within Tau amino acids 2 to 24 and binds monomericTau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau. Insome embodiments, an antibody binds an epitope of human Tau having, orconsisting of, the sequence AEPRQEFEVMEDHAGTYGLGDRK (SEQ ID NO: 2). Insome embodiments, an antibody binds an epitope of cynomolgus monkey Tauhaving, or consisting of, the sequence AEPRQEFDVMEDHAGTYGLGDRK (SEQ IDNO: 4). In some embodiments, an antibody binds an epitope of human Tauhaving, or consisting of, the sequence AEPRQEFEVMEDHAGTYGLGDRK (SEQ IDNO: 2) and an epitope of cynomolgus monkey Tau having, or consisting of,the sequence AEPRQEFDVMEDHAGTYGLGDRK (SEQ ID NO: 4).

In some embodiments, an antibody provided herein binds to an epitopewithin amino acids 19 to 33, 19 to 42, 37 to 51, 100 to 114, 118 to 132,or 172 to 177 of mature human Tau. In some embodiments, an antibody ofthe invention binds to an epitope within amino acids 19 to 33, 19 to 42,37 to 51, 100 to 114, 118 to 132, or 172 to 177 of mature human Tau andbinds monomeric Tau, oligomeric Tau, non-phosphorylated Tau, andphosphorylated Tau.

Antibodies of the invention are useful, e.g., for the diagnosis ortreatment of neurodegenerative diseases.

A. Exemplary Anti-Tau Antibodies

In some embodiments, the antibody comprises HVR-H1 comprising an aminoacid sequence of SEQ ID NO: 605; HVR-H2 comprising an amino acidsequence of SEQ ID NO: 606; and HVR-H3 comprising an amino acid sequenceof SEQ ID NO: 607.

In some embodiments, the antibody comprises HVR-L1 comprising an aminoacid sequence of SEQ ID NO: 608; HVR-L2 comprising an amino acidsequence of SEQ ID NO: 609; and HVR-L3 comprising an amino acid sequenceof SEQ ID NO: 610.

In some embodiments, the antibody comprises HVR-H1 comprising an aminoacid sequence of SEQ ID NO: 605; HVR-H2 comprising an amino acidsequence of SEQ ID NO: 606; HVR-H3 comprising an amino acid sequence ofSEQ ID NO: 607; HVR-L1 comprising an amino acid sequence of SEQ ID NO:608; HVR-L2 comprising an amino acid sequence of SEQ ID NO: 609; andHVR-L3 comprising an amino acid sequence of SEQ ID NO: 610.

In any of the above embodiments, an anti-Tau antibody is humanized. Insome embodiments, an anti-Tau antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-Tau antibody comprises a heavy chain variabledomain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 340, 603, 614, or 619. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-Tau antibody comprising that sequence retains the ability to bindto Tau. In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 340, 603, 614, or619. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-Tau antibody comprises the VH sequence in SEQ ID NO: 340, 603, 614,or 619, including post-translational modifications of that sequence. Ina particular embodiment, the VH comprises one, two or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 605, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:606, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:607.

In another aspect, an anti-Tau antibody comprises a light chain variabledomain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 341, 604, 615, or 620. In certain embodiments, a VL sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-Tau antibody comprising that sequence retains the ability to bindto Tau. In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 341, 604, 615, or620. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-Tau antibody comprises the VL sequence in SEQ ID NO: 341, 604, 615,or 620, including post-translational modifications of that sequence. Ina particular embodiment, the VL comprises one, two or three HVRsselected from: (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 608, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:609, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:610.

In some embodiments, an anti-Tau antibody comprises:

-   -   a) a heavy chain variable region (VH) comprising a sequence that        is at least 95% identical to SEQ ID NO: 603;    -   b) a light chain variable region (VL) comprising a sequence that        is at least 95% identical to SEQ ID NO: 604;    -   c) a VH as in (a) and a VL as in (b);    -   d) a heavy chain variable region (VH) comprising a sequence that        is at least 95% identical to SEQ ID NO: 614;    -   e) a light chain variable region (VL) comprising a sequence that        is at least 95% identical to SEQ ID NO: 615;    -   f) a VH as in (d) and a VL as in (e);    -   g) a heavy chain variable region (VH) comprising a sequence that        is at least 95% identical to SEQ ID NO: 619;    -   h) a light chain variable region (VL) comprising a sequence that        is at least 95% identical to SEQ ID NO: 620;    -   i) a VH as in (g) and a VL as in (h).

In some embodiments, an anti-Tau antibody comprises:

-   -   a) a heavy chain variable region (VH) comprising SEQ ID NO: 603;    -   b) a light chain variable region (VL) comprising SEQ ID NO: 604;    -   c) a VH as in (a) and a VL as in (b);    -   d) a heavy chain variable region (VH) comprising the sequence of        SEQ ID NO: 614;    -   e) a light chain variable region (VL) comprising the sequence of        SEQ ID NO: 615;    -   f) a VH as in (d) and a VL as in (e);    -   g) a heavy chain variable region (VH) comprising the sequence of        SEQ ID NO: 619;    -   h) a light chain variable region (VL) comprising the sequence of        SEQ ID NO: 620;    -   i) a VH as in (g) and a VL as in (h).

In some embodiments, an anti-Tau antibody comprises a heavy chainvariable region comprising a sequence selected from SEQ ID NOs: 603,614, and 619; and a light chain variable region comprising a sequenceselected from SEQ ID NOs: 604, 615, and 620. In some embodiments, theantibody comprises a heavy chain variable region comprising a sequenceselected from SEQ ID NOs: 340, 603, 614, and 619; and a light chainvariable region comprising a sequence selected from SEQ ID NOs: 604,615, and 620. In some embodiments, the antibody comprises a heavy chainvariable region comprising a sequence selected from SEQ ID NOs: 603,614, and 619; and a light chain variable region comprising a sequenceselected from SEQ ID NOs: 341, 604, 615, and 620.

In some embodiments, an anti-Tau antibody comprises (a) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 603 anda light chain variable region comprising the amino acid sequence of SEQID NO: 604; (b) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 614 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 615; or (c) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 619 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:620.

In some embodiments, an anti-Tau antibody comprises (a) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612and a light chain comprising the amino acid sequence of SEQ ID NO: 613;(b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 616or SEQ ID NO: 617 and a light chain comprising the amino acid sequenceof SEQ ID NO: 618; or (c) a heavy chain comprising the amino acidsequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a light chaincomprising the amino acid sequence of SEQ ID NO: 623.

In some embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a lightchain comprising the amino acid sequence of SEQ ID NO: 613. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 611 and a light chain comprising the amino acidsequence of SEQ ID NO: 613. In some embodiments, an isolated antibodythat binds to human Tau is provided, wherein the antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 612 and alight chain comprising the amino acid sequence of SEQ ID NO: 613. Insome embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a lightchain consisting of the amino acid sequence of SEQ ID NO: 613. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain consisting of the aminoacid sequence of SEQ ID NO: 611 and a light chain consisting of theamino acid sequence of SEQ ID NO: 613. In some embodiments, an isolatedantibody that binds to human Tau is provided, wherein the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 612 and a light chain consisting of the amino acid sequence of SEQID NO: 613.

In some embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 616 or 617 and a light chaincomprising the amino acid sequence of SEQ ID NO: 618. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 616 and a light chain comprising the amino acidsequence of SEQ ID NO: 618. In some embodiments, an isolated antibodythat binds to human Tau is provided, wherein the antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 617 and alight chain comprising the amino acid sequence of SEQ ID NO: 618. Insome embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 616 or SEQ ID NO: 617 and a lightchain consisting of the amino acid sequence of SEQ ID NO: 618. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain consisting of the aminoacid sequence of SEQ ID NO: 616 and a light chain consisting of theamino acid sequence of SEQ ID NO: 618. In some embodiments, an isolatedantibody that binds to human Tau is provided, wherein the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 617 and a light chain consisting of the amino acid sequence of SEQID NO: 618.

In some embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a lightchain comprising the amino acid sequence of SEQ ID NO: 623. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 621 and a light chain comprising the amino acidsequence of SEQ ID NO: 623. In some embodiments, an isolated antibodythat binds to human Tau is provided, wherein the antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 622 and alight chain comprising the amino acid sequence of SEQ ID NO: 623. Insome embodiments, an isolated antibody that binds to human Tau isprovided, wherein the antibody comprises a heavy chain consisting of theamino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a lightchain consisting of the amino acid sequence of SEQ ID NO: 623. In someembodiments, an isolated antibody that binds to human Tau is provided,wherein the antibody comprises a heavy chain consisting of the aminoacid sequence of SEQ ID NO: 621 and a light chain consisting of theamino acid sequence of SEQ ID NO: 623. In some embodiments, an isolatedantibody that binds to human Tau is provided, wherein the antibodycomprises a heavy chain consisting of the amino acid sequence of SEQ IDNO: 622 and a light chain consisting of the amino acid sequence of SEQID NO: 623.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-Tau antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an antibody selected from 37D3-H9, hu37D3-H9.v28.A4,hu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93. In certain embodiments,an antibody is provided that binds to an epitope within a fragment ofTau consisting of amino acids 2-24 of SEQ ID NO: 2. In certainembodiments, an antibody is provided that binds to an epitope within afragment of Tau consisting of amino acids 7-24 of SEQ ID NO: 2. Incertain embodiments, an antibody is provided that binds to an epitopewithin a fragment of Tau consisting of amino acids 7-20 of SEQ ID NO: 2.In certain embodiments, an antibody is provided that binds to an epitopewithin a fragment of Tau consisting of amino acids 10-24 of SEQ ID NO:2. In certain embodiments, an antibody is provided that binds to anepitope within a fragment of Tau consisting of amino acids 7-21 of SEQID NO: 2. In certain embodiments, an antibody is provided that binds toan epitope within a fragment of Tau consisting of amino acids 8-22 ofSEQ ID NO: 2. In certain embodiments, an antibody is provided that bindsto an epitope within a fragment of Tau consisting of amino acids 11-25of SEQ ID NO: 2. In certain embodiments, an antibody is provided thatbinds to one or more, or all, of the following fragments of Tau: 2-24,7-24, 7-20, 10-24, 7-21, 8-22, and 11-25. In some embodiments, anantibody is provided that binds to a peptide having the sequence of SEQID NO: 593, but does not bind to a peptide having the sequence of SEQ IDNO: 596 or SEQ ID NO: 597.

In a further aspect of the invention, an anti-Tau antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-Tauantibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 or IgG4 antibody or other antibodyclass or isotype as defined herein.

In a further aspect, an anti-Tau antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³M).

In some embodiments, K_(D) is measured by a radiolabeled antigen bindingassay (RIA). In some embodiments, an RIA is performed with the Fabversion of an antibody of interest and its antigen. For example,solution binding affinity of Fabs for antigen is measured byequilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigenin the presence of a titration series of unlabeled antigen, thencapturing bound antigen with an anti-Fab antibody-coated plate (see,e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establishconditions for the assay, MICROTITER® multi-well plates (ThermoScientific) are coated overnight with 5 μg/ml of a capturing anti-Fabantibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), andsubsequently blocked with 2% (w/v) bovine serum albumin in PBS for twoto five hours at room temperature (approximately 23° C.). In anon-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, K_(D) is measured using a BIACORE®surface plasmon resonance assay. For example, an assay using aBIACORE®-2000 or a BIACORE e-3000 (BIAcore, Inc., Piscataway, N.J.) isperformed at 25° C. or 37° C. with immobilized antigen CM5 chips at −10resonance units (RU). In some embodiments, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NETS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 resonance units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (K_(D)) is calculated as the ratio k_(off)/k_(on). See, e.g.,Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, then theon-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B 1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE′technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J Mol. Biol.338(2): 299-310 (2004); Lee et al., J Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for Tau and the other is for any other antigen. Incertain embodiments, one of the binding specificities is for Tau and theother is for amyloid beta. In certain embodiments, bispecific antibodiesmay bind to two different epitopes of Tau. Bispecific antibodies mayalso be used to localize cytotoxic agents to cells which express Tau.Bispecific antibodies can be prepared as full length antibodies orantibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to Tau as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In some embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (e. g. complex,hybrid and high mannose structures) as measured by MALDI-TOF massspectrometry, as described in WO 2008/077546, for example. Asn297 refersto the asparagine residue located at about position 297 in the Fc region(Eu numbering of Fc region residues); however, Asn297 may also belocated about ±3 amino acids upstream or downstream of position 297,i.e., between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, or434, e.g., substitution of Fc region residue 434 (U.S. Pat. No.7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In some embodiments, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In some embodiments,isolated nucleic acid encoding an anti-Tau antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In some embodiments, thehost cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell orlymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, a methodof making an anti-Tau antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-Tau antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-Tau antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with an antibody described herein for binding toTau. In certain embodiments, such a competing antibody binds to the sameepitope (e.g., a linear or a conformational epitope) that is bound by37D3-H9, hu37D3-H9.v28.A4, hu37D3-H9.v76, hu37D3-H9.v83, orhu37D3-H9.v93. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.).

In an exemplary competition assay, immobilized Tau (such as monomericTau) is incubated in a solution comprising a first labeled antibody thatbinds to Tau (e.g., any antibody described herein, such ashu37D3-H9.v28.A4) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to Tau.The second antibody may be present in a hybridoma supernatant. As acontrol, immobilized Tau is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to Tau,excess unbound antibody is removed, and the amount of label associatedwith immobilized Tau is measured. If the amount of label associated withimmobilized Tau is substantially reduced in the test sample relative tothe control sample, then that indicates that the second antibody iscompeting with the first antibody for binding to Tau. See Harlow andLane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-Tau (e.g.,pan-Tau) antibodies thereof having biological activity. Biologicalactivity may include, e.g., binding of such antibodies to multiple formsof Tau (e.g., monomeric Tau, oligomeric Tau, non-phosphorylated Tau, andphosphorylated Tau) and reducing the level of Tau protein (e.g., totalTau, total soluble Tau, soluble non-phosphorylated Tau, solublephosphorylated Tau, total insoluble Tau, insoluble non-phosphorylatedTau, insoluble phosphorylated Tau, hyperphosphorylated Tau, or pairedhelical filaments containing hyperphosphorylated Tau, in the brain,e.g., in the brain cortex and/or hippocampus). Antibodies having suchbiological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity. For example, an animal model of tauopathy, such asa Tau transgenic mice (e.g., P301L), can be used to detect binding ofanti-Tau antibodies to brain sections, and for example, toneurofibrillary tangles in the brains of the transgenic mice. Further,an animal model of tauopathy, such as a Tau transgenic mice (e.g.,P301L), can be treated with anti-Tau antibodies and experimentaltechniques known in the art can be used to assess whether such treatmentreduces the level of Tau protein (e.g., total Tau, total soluble Tau,soluble phosphorylated Tau, soluble non-phosphorylated Tau, totalinsoluble Tau, insoluble phosphorylated Tau, insolublenon-phosphorylated Tau, hyperphosphorylated Tau, or paired helicalfilaments containing hyperphosphorylated Tau) in the mouse brain (e.g.,in the brain cortex and/or hippocampus).

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-Tauantibody herein conjugated to one or more other therapeutic agents orradioactive isotopes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or 1123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody may be made using a variety of bifunctionalprotein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctionalderivatives of imidoesters (such as dimethyl adipimidate HCl), activeesters (such as disuccinimidyl suberate), aldehydes (such asglutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinyl sulfone)benzoate) which are commerciallyavailable (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-Tau antibodies provided hereinis useful for detecting the presence of Tau in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue, such as cerebrospinal fluid, a cell or tissue of the brain(e.g., brain cortex or hippocampus), or blood. In some embodiments, abiological sample is cerebrospinal fluid.

In some embodiments, an anti-Tau antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of Tau in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-Tau antibody as described herein under conditionspermissive for binding of the anti-Tau antibody to Tau, and detectingwhether a complex is formed between the anti-Tau antibody and Tau. Suchmethod may be an in vitro or in vivo method. Further, the complex formedbetween the anti-Tau antibody and Tau in a test biological sample can becompared to the complex formed in a control biological sample (e.g., abiological sample from a healthy subject or subjects). The amount of thecomplex formed between the anti-Tau antibody and Tau in a testbiological sample can also be quantified and compared to the amount ofthe complex formed in a control biological sample (e.g., a biologicalsample from a healthy subject or subjects) or to the average amount ofthe complex known to be formed in healthy subjects.

In some embodiments, an anti-Tau antibody is used to select subjectseligible for therapy with an anti-Tau antibody, e.g. where Tau is abiomarker for selection of patients. For example, in some embodiments,an anti-Tau (e.g., pan-Tau) antibody is used to detect whether thesubject has a Tau protein disease or disorder, or whether the subject isat high risk (or predisposed to) a Tau protein disease or disorder.

Exemplary diseases or disorders that may be diagnosed using an antibodyof the invention include Tau protein associated diseases or disorders,and diseases or disorders caused by or associated with the formation ofneurofibrillary tangles or neuropil threads. In some embodiments,diseases or disorders that may be diagnosed using an antibody of theinvention include Tau protein associated diseases or disorders that aremanifested in an impairment or loss of cognitive functions includingreasoning, situational judgement, memory capacity, learning, and/orspecial navigation. In particular, diseases or disorders that may bediagnosed using an antibody of the invention include tauopathies such asneurodegenerative tauopathies. Exemplary diseases or disorders that maybe diagnosed using an antibody of the invention include, but are notlimited to, Alzheimer's Disease, Creutzfeldt-Jacob disease, Dementiapugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease,inclusion-body myositis, prion protein cerebral amyloid angiopathy,traumatic brain injury, amyotrophic lateralsclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motorneuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangleswith calcification, frontotetemporal dementia, frontotemporal dementiawith parkinsonism linked to chromosome 17, Hallevorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy. Further nonlimiting exemplary diseases anddisorders that may be diagnosed using an antibody of the inventioninclude PART (primary age-related Tauopathy), tangle predominantdementia, subacute sclerosis panencephalopathy, chronic traumaticencephalopathy (CTE), white matter tauopathy with globular glialinclusions, Lewy body dementia (LBD), mild cognitive impairment (MCI),glaucoma, familial British dementia, familiar Danish dementia,Guadeloupean Parkinsonism, neurodegeneration with brain ironaccumulation, SLC9A6-related mental retardation, multiple sclerosis,HIV-related dementia, senile cardiac amyloidosis, and Huntington'sdisease. In some embodiments, a disorder that may be diagnosed using anantibody of the invention is Alzheimer's Disease (AD).

In certain embodiments, labeled anti-Tau antibodies are provided. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-Tau antibody as described hereinare prepared by mixing such antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers,diluents, and/or excipients (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)), in the form of lyophilized formulationsor aqueous solutions. Pharmaceutically acceptable carriers, diluents,and excipients are generally nontoxic to recipients at the dosages andconcentrations employed, and include, but are not limited to: sterilewater, buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-Tau antibodies provided herein may be used intherapeutic methods.

In one aspect, an anti-Tau antibody for use as a medicament is provided.In further aspects, an anti-Tau antibody for use in treating a Tauprotein associated disease or disorder is provided. In some embodiments,an anti-Tau antibody for use in treating diseases or disorders caused byor associated with the formation of neurofibrillary tangles or neuropilthreads is provided. In particular embodiments, an anti-Tau antibody foruse in treating a tauopathy such as a neurodegenerative tauopathy isprovided. Exemplary Tau protein associated diseases or disorders thatcan be treated that can be treated with anti-tau antibodies include,without limitation, Alzheimer's Disease, amyotrophic lateral sclerosis,Parkinson's disease, Creutzfeldt-Jacob disease, Dementia pugilistica,Down's Syndrome, Gerstmann-Straussler-Scheinker disease, inclusion-bodymyositis, prion protein cerebral amyloid angiopathy, traumatic braininjury, amyotrophic lateral sclerosis/parkinsonism-dementia complex ofGuam, Non-Guamanian motor neuron disease with neurofibrillary tangles,argyrophilic grain dementia, corticobasal degeneration, diffuseneurofibrillary tangles with calcification, frontotetemporal dementia,frontotemporal dementia with parkinsonism linked to chromosome 17,Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick diseasetype C, Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy. Further exemplary Tau protein associateddiseases or disorders that can be treated that can be treated withanti-tau antibodies include, without limitation, PART (primaryage-related Tauopathy), tangle predominant dementia, subacute sclerosispanencephalopathy, chronic traumatic encephalopathy (CTE), white mattertauopathy with globular glial inclusions, Lewy body dementia (LBD), mildcognitive impairment (MCI), glaucoma, familial British dementia,familiar Danish dementia, Guadeloupean Parkinsonism, neurodegenerationwith brain iron accumulation, SLC9A6-related mental retardation,multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis,and Huntington's disease. In some embodiments, an anti-Tau antibody foruse in treating Alzheimer's Disease (AD) is provided herein. In someembodiments, an anti-Tau antibody for use in treating moderate AD, mildto moderate AD, mild AD, early AD, or prodromal AD is provided herein.Further, Tau protein associated diseases or disorders that can betreated with an anti-Tau antibody include diseases or disorders that aremanifested in an impairment or loss of a cognitive function such asreasoning, situational judgement, memory capacity, learning, and/orspecial navigation. In certain embodiments, an anti-Tau antibody for usein a method of treatment is provided. In certain embodiments, theinvention provides an anti-Tau antibody for use in a method of treatingan individual, having any one of the Tau associated diseases ordisorders described above, comprising administering to the individual aneffective amount of the anti-Tau antibody. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow.

In some embodiments, the antibody of the invention is used to treat anindividual having an MMSE score of between 20 and 30, between 20 and 26,between 24 and 30, between 21 and 26, between 22 and 26, between 22 and28, between 23 and 26, between 24 and 26, or between 25 and 26. In someembodiments, the patient has an MMSE score between 22 and 26. As usedherein, an MMSE score between two numbers includes the numbers at eachend of the range. For example, an MMSE score between 22 and 26 includesMMSE scores of 22 and 26.

In some embodiments, the antibodies of the invention are used to treatan individual who is ‘tau positive,’ e.g., a patient having brain taudeposits that are typical of Tau protein associated disorders, e.g., apatient having a positive Tau PET scan.

In further embodiments, the invention provides an anti-Tau antibody foruse in reducing the levels of Tau protein (e.g., total Tau, totalsoluble Tau, soluble phosphorylated Tau, total insoluble Tau, insolublephosphorylated Tau, hyperphosphorylated Tau, or paired helical filamentscontaining hyperphosphorylated Tau) in an individual. For example suchreduction can occur in the brain (e.g., in the brain cortex and/orhippocampus). In some embodiments, the invention provides an anti-Tauantibody for use in reducing the levels of phosphorylated Tau. In someembodiments, the invention provides an anti-Tau antibody for use inreducing the levels of insoluble Tau (e.g., insoluble phosphorylatedTau). In some embodiments, the invention provides an anti-Tau antibodyfor use in reducing the levels of hyperphosphorylated Tau. In someembodiments, the invention provides an anti-Tau antibody for use inreducing the levels of paired helical filaments (e.g., paired helicalfilaments containing hyperphosphorylated Tau) in a brain tissue (e.g.,in the brain cortex and/or hippocampus). In certain embodiments, theinvention provides an anti-Tau antibody for use in a method of reducingthe levels of Tau protein (e.g., total Tau, total soluble Tau, solublephosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau,hyperphosphorylated Tau, or paired helical filaments containinghyperphosphorylated Tau) in the brain (e.g., in the brain cortex and/orhippocampus) in an individual comprising administering to the individualan effective amount of the anti-Tau antibody to reduce the levels of Tauprotein. An “individual” according to any of the above embodiments is amammal, preferably a human.

In some embodiments, the invention provides an anti-Tau antibody for usein modulating the levels of Tau protein (e.g., total Tau, total solubleTau, soluble phosphorylated Tau, total insoluble Tau, insolublephosphorylated Tau, hyperphosphorylated Tau, or paired helical filamentscontaining hyperphosphorylated Tau), for example, in the brain (e.g., inthe brain cortex and/or hippocampus) of an individual.

In a further aspect, the invention provides for the use of an anti-Tauantibody in the manufacture or preparation of a medicament. In someembodiments, the medicament is for treatment of a Tau protein associateddisease or disorder. The Tau protein associated disease or disorder canbe a disease or disorders caused by or associated with the formation ofneurofibrillary tangles or neuropil threads. In particular embodiments,the medicament is for treatment of a tauopathy such as aneurodegenerative tauopathy. In specific embodiments, the medicament isfor treatment of diseases or disorders selected from the groupconsisting of: Alzheimer's Disease (AD), Creutzfeldt-Jacob disease,Dementia pugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinkerdisease, inclusion-body myositis, prion protein cerebral amyloidangiopathy, traumatic brain injury, amyotrophic lateralsclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motorneuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangleswith calcification, frontotetemporal dementia, frontotemporal dementiawith parkinsonism linked to chromosome 17, Hallevorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy. In some embodiments, the medicament is fortreatment of diseases or disorders selected from PART (primaryage-related Tauopathy), tangle predominant dementia, subacute sclerosispanencephalopathy, chronic traumatic encephalopathy (CTE), white mattertauopathy with globular glial inclusions, Lewy body dementia (LBD), mildcognitive impairment (MCI), glaucoma, familial British dementia,familiar Danish dementia, Guadeloupean Parkinsonism, neurodegenerationwith brain iron accumulation, SLC9A6-related mental retardation,multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis,and Huntington's disease. In some embodiments, the medicament is fortreatment of AD. In particular embodiments, the medicament is fortreatment of a Tau associated disease or disorder that is manifested inan impairment or loss of a cognitive function such as reasoning,situational judgement, memory capacity, learning, or special navigation.In a further embodiment, the medicament is for use in a method oftreating one of the above-listed diseases (e.g., a tauopathy such as AD)comprising administering to an individual having such disease aneffective amount of the medicament. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, e.g., as described below.

In a further embodiment, the medicament is for reducing the levels ofTau protein (e.g., total Tau, total soluble Tau, solublenon-phorphorylated Tau, soluble phosphorylated Tau, total insoluble Tau,insoluble phosphorylated Tau, insoluble non-phorphorylated Tau,hyperphosphorylated Tau, or paired helical filaments containinghyperphosphorylated Tau). For example, such reducing of Tau protein canbe observed in the brain (e.g., in the brain cortex and/or hippocampus)or in cerebrospinal fluid of an individual. In some embodiments, themedicament is for reducing the levels of paired helical filaments. In afurther embodiment, the medicament is for use in a method of reducingthe levels of Tau protein (e.g., total Tau, total soluble Tau, solublephosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau,hyperphosphorylated Tau, or paired helical filaments containinghyperphosphorylated Tau) in an individual comprising administering tothe individual an effective amount of the medicament to reducing thelevels of Tau protein. An “individual” according to any of the aboveembodiments is a mammal, preferably, a human.

In a further aspect, the invention provides a method for treating a Tauprotein associated disease or disorder. Tau protein associated diseaseor disorder that can be treated in accordance with the methods providedherein include diseases or disorders caused by or associated with theformation of neurofibrillary tangles or neuropil threads. In particularembodiments, the invention provides a method for treating a tauopathysuch as a neurodegenerative tauopathy. In specific embodiments, theinvention provides a method for treating a disease or disorder selectedfrom the group consisting of: Alzheimer's Disease, Creutzfeldt-Jacobdisease, Dementia pugilistica, Down's Syndrome,Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis, prionprotein cerebral amyloid angiopathy, traumatic brain injury, amyotrophiclateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanianmotor neuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangleswith calcification, frontotetemporal dementia, frontotemporal dementiawith parkinsonism linked to chromosome 17, Hallevorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy. In some embodiments, the invention providesmethods for treating diseases or disorders selected from PART (primaryage-related Tauopathy), tangle predominant dementia, subacute sclerosispanencephalopathy, chronic traumatic encephalopathy (CTE), white mattertauopathy with globular glial inclusions, Lewy body dementia (LBD), mildcognitive impairment (MCI), glaucoma, familial British dementia,familiar Danish dementia, Guadeloupean Parkinsonism, neurodegenerationwith brain iron accumulation, SLC9A6-related mental retardation,multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis,and Huntington's disease. In some embodiments, the invention provides amethod for treating Alzheimer's Disease (AD). In particular embodiments,the invention provides a method for treating a Tau protein associateddisease or disorder that is manifested in an impairment or loss of acognitive function such as reasoning, situational judgement, memorycapacity, learning, or special navigation. In some embodiments, themethod comprises administering to an individual, having any one of thediseases or disorders described above, an effective amount of ananti-Tau antibody. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. In someembodiments, the method comprises administering to an individual havingone of the diseases described herein an effective amount of an anti-Tauantibody. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, as described below. An “individual”according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for reducing thelevels of Tau protein (e.g., total Tau, total soluble Tau, solublephosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau,hyperphosphorylated Tau, or paired helical filaments containinghyperphosphorylated Tau) in an individual. For example, such reducing ofthe levels of Tau protein can be observed in the brain (e.g., braincortex and/or hippocampus) or cerebrospinal fluid of an individual. Insome embodiments, the invention provides a method for reducing thelevels of paired helical filaments. In some embodiments, the methodcomprises administering to the individual an effective amount of ananti-Tau antibody to reduce the levels of Tau protein. In someembodiments, an “individual” is a human.

In some aspects, the invention provides a method for alleviating one ormore symptoms of a Tau protein associated disease or disorder; or ananti-Tau antibody or a medicament comprising anti-Tau antibody foralleviating one or more symptoms of a Tau protein associated disease ordisorder (such as any of the diseases or disorders described herein, forexample, AD). In some aspects, the invention provides a method forreducing the number of symptoms or the severity of one or more symptomsof a Tau protein associated disease or disorder; or an anti-Tau antibodyor a medicament comprising anti-Tau antibody for reducing the number ofsymptoms or the severity of one or more symptoms of a Tau proteinassociated disease or disorder (such as any of the diseases or disordersdescribed herein, for example, AD). In a particular embodiment, thesymptom of a Tau protein associated disease or disorder is an impairmentin cognition. In a specific embodiment, the symptom of a Tau proteinassociated disease or disorder is an impairment in learning and/ormemory. In a specific embodiment, the symptom of a Tau proteinassociated disease or disorder is a long-term memory loss. In a specificembodiment, the symptom of a Tau protein associated disease or disorderis dementia. In some embodiments, the symptom of a Tau proteinassociated disease or disorder is confusion, irritability, aggression,mood swings, or a language impairment. In some embodiments, the symptomof a Tau protein associated disease or disorder is an impairment or lossof one or more cognitive functions such as reasoning, situationaljudgment, memory capacity, and/or learning. The methods provided hereincomprise administration of an amount (e.g., therapeutically effectiveamount) of an anti-Tau antibody to an individual (e.g., who displays oneor more symptoms of a Tau protein associated disease or disorder).

In specific aspects, the invention provides a method for retaining orincreasing cognitive memory capacity, or for slowing down memory lossassociated with a Tau protein associated disease or disorder; or ananti-Tau antibody or a medicament comprising anti-Tau antibody forretaining or increasing cognitive memory capacity or for slowing downmemory loss associated with a Tau protein associated disease or disorder(such as any of the diseases or disorders described herein, for example,AD). The methods provided herein comprise administration of an amount(e.g., therapeutically effective amount) of an anti-Tau antibody to anindividual (e.g., who displays one or more symptoms of memory loss or adecrease of memory capacity).

In some aspects, the invention provides a method for decreasing the rateof progression of a Tau protein associated disease or disorder; or ananti-Tau antibody or a medicament comprising anti-Tau antibody fordecreasing the rate of progression of a Tau protein associated diseaseor disorder (such as any of the diseases or disorders described herein,for example, AD). The methods provided herein comprise administration ofan amount (e.g., therapeutically effective amount) of an anti-Tauantibody to an individual (e.g., who displays one or more symptoms of aTau protein associated disease or disorder).

In some aspects, the invention provides a method for preventing thedevelopment of a Tau protein associated disease or disorder; or ananti-Tau antibody or a medicament comprising anti-Tau antibody forpreventing the development of a Tau protein associated disease ordisorder (such as any of the diseases or disorders described herein, forexample, AD). The methods provided herein comprise administration of anamount (e.g., therapeutically effective amount) of an anti-Tau antibodyto an individual (e.g., who is at risk of a Tau protein associateddisease or disorder).

In some aspects, the invention provides a method for delaying thedevelopment of a Tau protein associated disease or disorder; or ananti-Tau antibody or a medicament comprising anti-Tau antibody fordelaying the development of a Tau protein associated disease or disorder(such as any of the diseases or disorders described herein, for example,AD). The methods provided herein comprise administration of an amount(e.g., therapeutically effective amount) of an anti-Tau antibody to anindividual (e.g., who displays one or more symptoms of a Tau proteinassociated disease or disorder).

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-Tau antibodies provided herein, e.g., for usein any of the above therapeutic methods. In some embodiments, apharmaceutical formulation comprises any of the anti-Tau antibodiesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the anti-Tauantibodies provided herein and at least one additional therapeuticagent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent.

For example, the composition according to the invention may beadministered in combination with other compositions comprising anadditional therapeutic agent, such as a biologically active substance orcompound such as, for example, a known compound used in the medicationof tauopathies and/or of amyloidoses, a group of diseases and disordersassociated with amyloid or amyloid-like protein such as the amyloidprotein involved in Alzheimer's Disease.

Generally, the other biologically active compound may includeneuron-transmission enhancers, psychotherapeutic drugs, acetylcholineesterase inhibitors, calcium-channel blockers, biogenic amines,benzodiazepine tranquillizers, acetylcholine synthesis, storage orrelease enhancers, acetylcholine postsynaptic receptor agonists,monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamatereceptor antagonists, non-steroidal anti-inflammatory drugs,antioxidants, serotonergic receptor antagonists, or other therapeuticagents. In particular, the biologically active agent or compound maycomprise at least one compound selected from compounds against oxidativestress, anti-apoptotic compounds, metal chelators, inhibitors of DNArepair such as pirenzepine and metabolites, 3-amino-1-propanesulfonicacid (3APS), 1,3-propanedisulfonate (1,3PDS), secretase activators,beta- and gamma-secretase inhibitors, tau proteins, anti-Tau antibodies(including, but not limited to, antibodies disclosed in WO2012049570,WO2014028777, WO2014165271, WO2014100600, WO2015200806, U.S. Pat. Nos.8,980,270, and 8,980,271), neurotransmitter, beta-sheet breakers,antiinflammatory molecules, “atypical antipsychotics” such as, forexample clozapine, ziprasidone, risperidone, aripiprazole or olanzapineor cholinesterase inhibitors (ChEIs) such as tacrine, rivastigmine,donepezil, and/or galantamine and other drugs and nutritive supplementssuch as, for example, vitamin B 12, cysteine, a precursor ofacetylcholine, lecithin, choline, Ginkgo biloba, acyetyl-L-carnitine,idebenone, propentofylline, or a xanthine derivative, together with abinding peptide according to the invention including antibodies,particularly monoclonal antibodies and active fragments thereof, and,optionally, a pharmaceutically acceptable carrier and/or a diluentand/or an excipient and instructions for the treatment of diseases.

In some embodiments, an antibody of the invention may be administered incombination with a neurological drug. Such neurological drugs include,but are not limited to, an antibody or other binding molecule(including, but not limited to a small molecule, a peptide, an aptamer,or other protein binder) that specifically binds to a target selectedfrom: beta secretase, presenilin, amyloid precursor protein or portionsthereof, amyloid beta peptide or oligomers or fibrils thereof, deathreceptor 6 (DR6), receptor for advanced glycation endproducts (RAGE),parkin, and huntingtin; an NMDA receptor antagonist (i.e., memantine), amonoamine depletor (i.e., tetrabenazine); an ergoloid mesylate; ananticholinergic antiparkinsonism agent (i.e., procyclidine,diphenhydramine, trihexylphenidyl, benztropine, biperiden andtrihexyphenidyl); a dopaminergic antiparkinsonism agent (i.e.,entacapone, selegiline, pramipexole, bromocriptine, rotigotine,selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa,pergolide, tolcapone and amantadine); a tetrabenazine; ananti-inflammatory (including, but not limited to, a nonsteroidalanti-inflammatory drug (i.e., indomethicin and other compounds listedabove); a hormone (i.e., estrogen, progesterone and leuprolide); avitamin (i.e., folate and nicotinamide); a dimebolin; a homotaurine(i.e., 3-aminopropanesulfonic acid; 3APS); a serotonin receptor activitymodulator (i.e., xaliproden); an, an interferon, and a glucocorticoid orcorticosteroid. The term “corticosteroid” includes, but is not limitedto, fluticasone (including fluticasone propionate (FP)), beclometasone,budesonide, ciclesonide, mometasone, flunisolide, betamethasone andtriamcinolone. “Inhalable corticosteroid” means a corticosteroid that issuitable for delivery by inhalation. Exemplary inhalable corticosteroidsare fluticasone, beclomethasone dipropionate, budenoside, mometasonefuroate, ciclesonide, flunisolide, and triamcinolone acetonide.

In some embodiments, one or more anti-amyloid beta (anti-Abeta)antibodies may be administered with an anti-Tau antibody providedherein. Non-limiting examples of such anti-Abeta antibodies includecrenezumab, solanezumab, bapineuzumab, aducanumab, and BAN-2401 (Biogen,Eisai Co., Ltd.). In some embodiments, one or more beta-amyloidaggregation inhibitors may be administered with an anti-Tau antibodyprovided herein. Nonlimiting exemplary beta-amyloid aggregationinhibitors include ELND-005 (also referred to as AZD-103 orscyllo-inositol), tramiprosate, and PTI-80 (Exebryl-1®; ProteoTech). Insome embodiments, one or more BACE inhibitors may be administered withan anti-Tau antibody provided herein. Non-limiting examples of such BACEinhibitors include E-2609 (Biogen, Eisai Co., Ltd.), AZD3293 (also knownas LY3314814; AstraZeneca, Eli Lilly & Co.), MK-8931 (verubecestat), andJNJ-54861911 (Janssen, Shionogi Pharma). In some embodiments, one ormore Tau inhibitors may be administered with an anti-Tau antibodyprovided herein. Non-limiting examples of such Tau inhibitors includemethylthioninium, LMTX (also known as leuco-methylthioninium orTrx-0237; TauRx Therapeutics Ltd.), Rember™ (methylene blue ormethylthioninium chloride [MTC]; Trx-0014; TauRx Therapeutics Ltd), PBT2(Prana Biotechnology), and PTI-51-CH3 (TauPro™; ProteoTech). In someembodiments, one or more other anti-Tau antibodies may be administeredwith an anti-Tau antibody provided herein. Non-limiting examples of suchother anti-Tau antibodies include BMS-986168 (Bristol-Myers Squibb) andC2N-8E12 (AbbVie, C2N Diagnostics, LLC). In some embodiments, a generalmisfolding inhibitor, such as NPT088 (NeuroPhage Pharmaceuticals), maybe administered with an anti-Tau antibody provided herein.

In some embodiments, the composition according to the invention maycomprise niacin or memantine together with a chimeric antibody or ahumanized antibody according to the invention including antibodies,particularly monoclonal antibodies and active fragments thereof, and,optionally, a pharmaceutically acceptable carrier and/or a diluentand/or an excipient.

In some embodiments, compositions are provided that comprise “atypicalantipsychotics” such as, for example clozapine, ziprasidone,risperidone, aripiprazole or olanzapine for the treatment of positiveand negative psychotic symptoms including hallucinations, delusions,thought disorders (manifested by marked incoherence, derailment,tangentiality), and bizarre or disorganized behavior, as well asanhedonia, flattened affect, apathy, and social withdrawal, togetherwith the chimeric antibody or the humanized antibody according to theinvention or active fragments thereof, and, optionally, apharmaceutically acceptable carrier and/or a diluent and/or anexcipient.

Other compounds that can be suitably used in compositions in addition tochimeric antibody or humanized antibody according to the invention, arethose disclosed, for example, in WO 2004/058258 (see especially pages 16and 17) including therapeutic drug targets (page 36-39), alkanesulfonicacids and alkanolsulfuric acid (pages 39-51), cholinesterase inhibitors(pages 51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages58-59), non-steroidal anti-inflammatory drugs (pages 60-61),antioxidants (pages 61-62), peroxisome proliferators-activated receptors(PPAR) agonists (pages 63-67), cholesterol-lowering agents (pages68-75); amyloid inhibitors (pages 75-77), amyloid formation inhibitors(pages 77-78), metal chelators (pages 78-79), anti-psychotics andanti-depressants (pages 80-82), nutritional supplements (pages 83-89)and compounds increasing the availability of biologically activesubstances in the brain (see pages 89-93) and prodrugs (pages 93 and94), which document is incorporated herein by reference, but especiallythe compounds mentioned on the pages indicated above.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In some embodiments, administration of theanti-Tau antibody and administration of an additional therapeutic agentoccur within about one month, or within about one, two or three weeks,or within about one, two, three, four, five, or six days, of each other.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-Tau antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-Tau antibody.

III. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1: Generation of Tau for Immunization Generation of MonomericRecombinant Tau

The recombinant human Tau construct, 2N4R isoform (amino acids 2-441),was fused to a N-terminal His-tag to facilitate purification andcharacterization. See, e.g., FIG. 15. The fusion construct was clonedinto the pET52b vector (Novagen) and expressed in E. coli. Cells wereharvested and lysed under denaturing condition using 7M guanidiniumchloride overnight at 4° C. with stirring. Cell debris was pelleted at40,000 rpm for 1 hour. The recombinant, His-tagged protein was isolatedby nickel affinity chromatography (Ni Sepharose excel affinity resin, GEHealthcare Life Sciences) followed by size-exclusion chromatography(Superdex 200 resin, GE Healthcare Life Sciences) under denaturingcondition. Guanidinium chloride was removed by dialyzing the recoveredprotein into 20 mM MES, 50 mM NaCl, and 1 mM TCEP at pH 6.8. The His-tagwas subsequently removed using TEV protease, followed by finalpurification using cation exchange chromatography (Mono S column, GEHealthcare Life Sciences) to remove the cleaved His-tag. Thepurification buffer contained 0.1% Triton x-114 (v/v) to removeendotoxin. Purified protein was exchanged into PBS with 1 mM TCEP. Thepurity and monomeric state were analyzed by SDS-PAGE and SEC-MALLS.Identity was confirmed by mass spectrometry. Protein concentration wasdetermined by UV absorption at 280 nm. The final product was free ofendotoxin (<0.5 EU/mg), as determined by Kinetic Limulus AmebocyteLysate (LAL) assay.

Generation of Phosphorylated Tau

Phosphorylated Tau was generated using the Tau 2-441 construct preparedusing the method described above. The protein construct wasphosphorylated using 0.5 μM PKA kinase (Life Technologies), whichphosphorylates serine 409, among other residues. The reaction mixturewas incubated with 1 mM ATP, 5 mM MgCl₂, at room temperature for 72hours. Phosphorylation was confirmed by mass spectrometry.Size-exclusion chromatography (Superdex 75, GE Healthcare Life Sciences)was used to remove the kinase. The purity, monomeric state, andendotoxin level of the phosphorylated protein preparation were analyzedsubstantially as described above.

In Vitro Oligomeriztion of Monomeric Tau

Oligomeric Tau was generated using the monomeric Tau 2-441 construct.The monomeric protein was first exchanged into 20 mMN,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 25 mM NaCl, pH7.4, followed by oligomeriztion using 75 μM arachidonic acid (CaymanChemicals) and 18 kDa Heparin (Sigma Aldrich), at equimolarconcentration with protein at 37° C. for 3 days. Oligomerization wasconfirmed by thioflavin T fluorescence assay, dynamic light scattering(DLS), and analytical size-exclusion chromatography. Oligomeric Tau isin some instances referred to as “oligoTau.”

Example 2: Generation of Anti-Tau Antibodies Methods Generation ofHybridomas

Female C57BL/6JOlaHsd (C57BL/6) and BALB/c OlaHsd (Balb/c) wild-typemice (Harlan, USA) were received at 9 weeks of age. Tau knock-out mice(B6.129-Mapttm1Hnd/J; The Jackson Laboratory, USA) were received at 6and 9 weeks of age. Vaccinations started at 12 to 15 weeks of age. Micewere vaccinated with oligomerized human Tau. Before vaccination, theoligoTau was mixed with one of two adjuvants used in this study, RibiAdjuvant System (Ribi; Sigma-Aldrich, Switzerland) at 50% v/v, or acombination of CpG single-stranded synthetic DNA oligodeoxynucleotides(CpG; Microsynth, Switzerland) and aluminium hydroxide (Al; Brenntag,Switzerland). Ribi is 2% squalene oil-in-water emulsion containingmonophosphoryl lipid A (isolated from Salmonella minnesota) andsynthetic trehalose dicorynomycolate (isolated from the cord factor ofthe Tubercle bacillus) in squalene oil, 0.2% Tween-80, and water.

Mice were vaccinated by subcutaneous injection (s.c.), except groups Dand G, which received a combination of intraperitoneal (i.p.) and hockadministrations. Mice in group D were administered 50 μg of oligoTaui.p. and 10 μg of oligoTau as hock injection. Mice in group G wereadministered 8 μg of oligoTau i.p. and 2 μg of oligoTau as hockinjection. See Table 2.

For vaccinations containing CpG and Al (CpG/Al) as adjuvant, eachinjection of 200 μL contained 60 μg (30 nmol) CpG, 1 mg Al, and 50 μgoligoTau. For all study groups, mice were injected on days 0, 14, 35,and 56. Mice used for myeloma fusion (Nanotools, Germany) wereadditionally vaccinated with three daily booster injections of oligoTau(50 μg per i.p. injection) without adjuvant added.

TABLE 2 Mice and vaccination protocols Total oligoTau dose VaccinationStudy group Mouse strain (μg/injection) Adjuvant route A C57BL/6 50CpG/AI s.c. B C57BL/6 50 Ribi s.c. C Balb/c 50 CpG/AI s.c. D Balb/c 60CpG/AI hock + i.p. E Balb/c 5 CpG/AI s.c. F Balb/c 50 Ribi s.c. G Tauknock-out 10 Ribi hock + i.p.

Mice were bled and sacrificed one day following the last of threebooster injections, and splenocytes were fused with myeloma cells togenerate antibody producing hybridomas.

Selection of Hybridomas for Subcloning

For fusions, mice were divided into three groups, for a total of 10fusions (2 fusions in one group, four fusions in the second group, andfour fusions in the third group), generating 299 hybridomas. Viablehybridomas were grown using serum-containing selection media, and thebest hybridomas were then selected for subcloning, using ELISA assaysfor full-length human Tau and oligoTau binding as described below.Following limiting dilution, the final hybridomas were then grown inserum-free medium and media was collected from stable colonies forantibody screening and selection.

ELISA Screening Assays

Serum-free supernatants were harvested from stable hybridomas. Thesupernatants containing antibodies of interest were then screened byELISA assays to characterize antibody properties and select antibodiesfor further development. The ELISA assays were used to determine thefollowing: binding to full-length human Tau (flTau; SignalChem, Canada),binding to hyperphosphorylated flTau (Genentech, USA), binding tooligomeric versus monomeric preparations of flTau, and binding tocertain antibody Tau epitope(s). Briefly, 96-well MaxiSorp ELISA plates(Nunc, Denmark) were coated with one of the targets as shown in Table 3.

TABLE 3 Targets used for the ELISA screening assays. Assay ELISA setupTarget Binding to flTau Direct ELISA Full-length human Tau (flTau)coated at 1 μg/mL Binding to pTau Direct ELISA Full-length human Tauphosphorylated in vitro using 4 kinases (GSK3β, Cdk5, PKA, and CK1δ;hyper- phosphorylated Tau or pTau) purified and coated at 1 μg/mLEpitope mapping Direct ELISA Biotinylated 15-mer peptides spanning the441 amino acids (aa) of human Tau with 9 aa offset and 6 aa overlapcoated at 10 μg/mL on a streptavidin 96-well plate Binding to CaptureAVI-tag biotinylated oligomeric and monomeric flTau captured in oligoTauELISA solution by anti-IgG immobilized antibodies being tested

Coating was done overnight in phosphate-buffered saline (PBS) at 4° C.Plates were washed thoroughly with 0.05% Tween-20/PBS and then blockedwith 1% bovine serum albumin (BSA) in 0.05% Tween-20/PBS for 1 hr at 37°C. The antibody contained in the hybridoma supernatant was then added atthe indicated dilutions, and incubated for 2 hrs at 37° C. after whichthe plates were washed as described previously.

For the direct ELISAs, an AP-conjugated anti-mouse IgG secondaryantibody (Jackson ImmunoResearch Laboratories, United Kingdom) was addedat 1/6000 dilution in 0.05% Tween-20/PBS for 2 hr at 37° C. After thefinal wash, plates were incubated with p-nitrophenyl phosphate disodiumhexahydrate (pNPP; Sigma-Aldrich, Switzerland) phosphatase substratesolution, and read at 405 nm using an ELISA plate reader (Tecan,Switzerland). Results are expressed as optical densities (O.D.).

For the oligoTau and monoTau capture ELISAs, antibodies contained inserum-free sterile hybridoma supernatants were immobilized on ananti-IgG coated plate at 500-fold dilution, followed by the incubationof oligoTau or monoTau, both with site-specific biotinylation via anAVI-tag. The target incubations started at 5 μg/mL and then were diluted8- or 16-fold. Streptavidin-HRP and ABTS substrate was used for signalquantitation in a plate reader (Tecan, Switzerland). Results areexpressed as O.D.

Affinity Estimates

Affinity of non-purified antibodies in serum-free hybridoma supernatantswas estimated by surface plasmon resonance using a Biacore T-100instrument (GE Healthcare, United Kingdom). Antibodies were immobilizedonto an anti-IgG biosensor chip, and flTau (SignalChem, Canada) was usedas the target analyte. Kinetic analysis was done using a 1:1 Langmuirfit model.

SDS-PAGE and Western-Blot Assays

The binding of selected panTau antibodies to Tau in human brain wastested in a Western-blot (WB) using brain lysates from three AD and twoage-matched non-AD control donors (Tissue Solutions, United Kingdom).The lysates were processed to obtain a detergent-free soluble Taufraction. Processed lysates were loaded onto 4-12% bis-tris gels (Novex,Life Technologies, Switzerland) and transferred onto Immobilon PVDFmembranes and blotted with antibodies being tested with and an IRDye800CW goat anti-mouse secondary antibody (Li-Cor, USA).

ELISA Assay Using Human Brain Lysates

To assess the binding of selected antibodies to non-denatured human Tauin AD and control brain lysates, antibodies from hybridoma supernatants,or a negative and positive control antibodies, were immobilized on a96-well plate as described above. Tau in soluble human brain lysatesfrom AD or age-matched control subjects (400m/mL protein; all fromTissue Solutions, United Kingdom) was then captured and detection wasperformed using a polyclonal rabbit panTau antibody (AbCam, UnitedKingdom) followed by an Fc-y fragment specific anti-rabbit IgG-AP(Jackson ImmunoResearch, USA). Brain lysate from Tau knock-out mouse wasused as a negative sample control. Plates were incubated with pNPP(Sigma-Aldrich) phosphatase substrate solution, and read at 405 nm usingan ELISA plate reader (Tecan, Switzerland). Results are expressed asoptical densities (O.D.).

Sequencing of Antibody Hybridomas

Hybridoma cell lysates were supplied to Antitope (Antitope, UnitedKingdom) for variable region gene sequencing. Briefly, RT-PCR wasperformed using degenerate primer pools for murine signal sequencestogether with constant region primers for each of IgG variable heavy(VH), IgM VH, Ig kappa variable light (KVL) and Ig λ VL chains. Heavychain V-region mRNA was amplified using a set of six degenerate primerpools (HA to HF) specific for VH signal sequences together with eitherIgM or IgG-specific constant region primers. The light chain V-regionmRNA was amplified using a set of eight signal sequence-specificdegenerate primer pools, seven for the κ cluster (KA to KG) and one forthe λ cluster (LA), together with either κ or λ constant region primers.The PCR products obtained from successful amplification were purified,cloned into a ‘TA’ cloning vector (pGEM-T Easy, Promega), transformedinto E. coli and individual colonies sequenced. The nucleotide and aminoacid sequences of the antibody VH and VL regions were determined withthe sequences for 27 antibody hybridomas.

Results Selection of Hybridomas for Subcloning

Hybridomas that were generated from each of the three rounds of fusions,a total of 299 hybridomas derived from ten fusions, were initiallyassayed for binding to flTau, with selected hybridomas additionallyassayed for binding to pTau and oligomerized Tau. The aim was to selectantibodies that bind equally well to Tau and to Tau modifiedpost-translationally, such as phosphorylated or oligomeric Tau. Forthis, assays were run on hybridomas to select for the best panTauproperties. To determine antibody binding region and the specific Tauepitope, the binding region was first determined using different Taufragments and then a library of 15-mer overlapping Tau peptides spanningthe full 441 amino acids (aa) sequence of the longest human Tau isoform.A group of antibodies binding to pre-determined regions of Tau wereintentionally avoided with the aim to maximize binding to differentpost-translationally modified forms of Tau and to all the six differenthuman Tau isoforms present in humans.

The three fusion series resulted in the generation of 133 subclonedstable hybridomas that were screened for the best panTau properties. Acombination of different screening assays was used to narrow down thenumber of antibody hybridomas having the preferred properties for apanTau antibody. For comparing flTau and pTau binding, 90 hybridomaswere assayed with the results of 24 hybridomas shown in FIG. 1A-F. As aninitial screen had been performed using Tau fragments to avoid selectingantibodies binding to regions of Tau known to have high density ofresidues that are phosphorylated in Alzheimer's disease (AD) and othertauopathies, most antibodies tested bound to both flTau and pTau withsimilar binding properties as determined by this ELISA.

In some embodiments, it is desirable that a panTau antibody bind to bothmonomeric and oligomeric forms of Tau without a strong preference to oneor the other. A capture ELISA was set up to determine if antibodiesbound to both monomeric and oligomeric forms of flTau. An ELISA run incapture mode preserves the oligomer conformation of pre-oligomerized Tauand the monomeric state of monoTau better than when run as a directELISA with the targets immobilized onto an ELISA plate.

Each assay was run by directly comparing the binding of the two forms ofTau to all 90 antibodies tested. Antibodies known to have preferredbinding to either oligoTau or that do not discriminate between the twoforms of Tau were used as controls in each assay. The results of 18hybridomas are shown in FIG. 2A-E.

Mapping the epitopes is important for selecting antibodies with goodpanTau properties, as antibodies that bind to regions with high densityof potential pTau residues (Ser, Thr, and Tyr) can be avoided. Bindingto all six isoforms of human Tau was also used as a selection criterionfor a panTau antibody. The panTau epitopes of antibodies that had beeninitially selected were verified and determined with improved accuracyusing a library of 49 peptides each having 15 amino acids (aa) spanningthe full length of human Tau, with an overlap of 6 aa residues and anoffset of 9 aa. The residue numbers are based on the longest isoform ofhuman Tau (441 aa). Non-purified antibdies were used at high 1/10dilution to verify binding versus no binding to all peptides. Screeningof antibodies from 112 hybridomas previously selected by ELISA indicatedbinding to 20 different Tau epitopes (Table 4).

TABLE 4 Tau epitopes for antibodies Antibody Tau epitope (aa) 14F5-D9 1-15 94B2-B12  1-15 94B2-C1  1-15 10A1-A6 10-24 10A1-D8 10-24 11E10-B810-24 17G12-C11 10-24 17G12-D5 10-24 19H6-A1 10-24 19H6-F7 10-24 19H6-G810-24 37D3-H12 10-24 37D3-H9 10-24 37E8-B4 10-24 37E8-C2 10-24 3A4-H410-24 3H10-E12(A) 10-24 3H10-G12 10-24 44B7-A9 10-24 44B7-B1 10-2454C1-H11 10-24 61E7-B11 10-24 61H10-B4 10-24 61H10-H3 10-24 127G7-A510-24 127G7-E7 10-24 115A4-A3 10-24 115A4-B1 10-24 125B11-B6 10-2473C8-A5 10-24 73C8-G4 10-24 76B4-D9 10-24 76B4-H7 10-24 123E9-B3 19-3315C6-A7 19-33 19F8-B1 19-33 24A11-D5 19-33 63H3-B2 19-33 63H3-D8 19-3364B9-E11 19-33 64B9-F12 19-33 45D2-C9 19-33 45D2-F4 19-33 72E12-B2 19-3372G10-A7 19-33 72G10-B6 19-33 123E9-A1 19-42 19F8-C11 19-42 7A11-C1219-42 89F4-A2 28-42 89F4-A1 28-44 12A10-E8 37-51 55E7-B12 37-51 72E12-H937-51 55E7-F11 37-51 30D12-B5 64-78 21C1-D8 64-78 21C1-G6 64-78 30D12-F664-78 31A3-A4 64-78 31A3-A7 64-78 77D1-D2 64-78 77D1-E6 64-78 30A1-C973-87 30A1-D11 73-87 28F5-G8 82-96 28F5-H8 82-96 33G9-A11 100-11433G9-B9 100-114 52F2-E12 100-114 52F2-E8 100-114 52F6-B3 100-11452F6-F11 100-114 56D3-C8 100-114 56D3-E9 100-114 70B10-B6B2 100-11470B10-B6G12 100-114 78E4-D11 100-114 78E4-G4 100-114 30G1-B2 109-12330G1-C11 109-123 49G10-F4 109-123 49G10-H1 109-123 65B1-A2 109-12365B1-A7 109-123 73H6-B8 109-123 113F5-A8 109-123 113F5-F7 109-123125B11-H3 109-123 26C1-B11 118-132 26C1-C8 118-132 74H10-A3 118-13274H10-C3 118-132 78F3-B2 118-132 78F3-E7C6 118-132 78F3-E7H7 118-132126H12-G6 136-150 126H12-H7 136-150 22G7-C9 154-168 22G7-G9 154-168111B8-C4 163-177 111B8-F10 163-177 66F5-A1 172-177 66F5-F2 172-17771H8-A1 190-204 71H8-D6 190-204 83E10-D10 190-204 83E10-D6 190-204126F11-B3 217-231 126F11-G11 217-231 93A8-C9 397-411 93A8-D2 397-411

For affinity measurements to flTau, 46 antibodies were measured usingSPR on a Biacore instrument, with the K_(D)s determined. Biacoreaffinity measurements were done by immobilizing antibodies on ananti-IgG chip and using flTau as the target analyte. Results for 32antibodies are shown in Table 5, with antibodies ranked based onaffinity to flTau. Of the antibodies measured for affinty to flTau, 22antibodies had affinities better than 20 nM, of which 14 antibodies hadK_(D)s under 5 nM with antibody 37D3-H9 having a K_(D) (affinity) of 1nM.

TABLE 5 Affinity for flTau K_(D) Antibody (nM) 37D3-H9 1 54C1-H11 1.5123E9-A1 1.8 94B2-C1 1.9 24A11-D5 2 113F5-F7 2.4 89F4-A1 2.9 19F8-B1 2.961E7-C4 3.3 126F11-G11 4.2 26C1-C8 4.3 93A8-D2 4.3 37E8-B4 4.4 61E7-6114.8 125B11-H3 6 54C1-C3 6.8 3A4-H4 7.8 52F6-F11 8.4 3A4-A12 10.1 44B7-B114.7 3H10-E12 19.4 10A1-D8 19.6 52F2-E8 26 19H6-F7 39 34H4-F5 43 19H6-A156 34H4-B10 69 17G12-C11 118 45H12-C4 139 17G12-D5 161 61H10-H3 17711E10-C3 399

To verify the binding of selected antibodies to all six isoforms ofhuman Tau, an SDS-PAGE was run with a recombinant Tau ladder containingall six isoforms and Western-blot (WB) done using three selected Tauantibodies. All three panTau antibodies bind to all six Tau isoforms(FIG. 3). Furthermore, brain homogenates from three AD and twoage-matched controls were simultaneously run for comparison. Asexpected, and based on the mapped epitopes, all three antibodies testedin this assay showed binding to all six Tau isoforms. The differenceobserved in band patterns between human AD and control donors mayrepresent the greater phosphorylation and/or SDS-stable Tau aggregatesthat would be expected to be present in AD subjects.

Human Alzheimer's disease (AD) and control samples were additionally runin a non-denaturing ELISA capture assay to verify binding to Tau inhuman brains. Samples lysates processed for soluble Tau from two AD andtwo non-AD age-matched control subjects were run at 8 dilutions testingthree antibodies (FIG. 4A-C).

Antibody variable chain sequences were determined for 27 hybridomas(Antitope, United Kingdom). Protein sequences for certain heavy andlight chain variable domains and hypervariable regions (HVRs) are shownin the Table of Sequences.

Example 3: Characterization Anti-Tau Antibodies

Antibody heavy and light chains were constructed via gene synthesis andsubcloning of the resulting DNA into murine IgG2a (heavy chain) andmurine kappa (light chain) mammalian expression vectors. Antibodies wereexpressed in CHO or 293T cells by transient co-transfection of the heavychain and light chain plasmids and were purified with affinity resinMabSelectSure (GE Healthcare Life Sciences). Purified recombinantantibodies were screened for binding to Tau monomer protein on a BiacoreT200 surface plasmon resonance instrument using a mouse IgG capture kitand a Series S CM5 chip. Antibodies in mIgG2a format diluted in 10 mMHEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (running buffer, HBSP) werecaptured for 30 or 45 seconds at a concentration of 1 μg/ml (antibodies26C1, 94B2-C1, 52F6-F11.v1, 52F6-F11.v2, 11E10-B8, 55E7-F11, 125B11-H3,123E9-A1, 30G1-B2, 66F5-A1, 89F4-A1, 93A8-D2 and 126F11-G11) or for 70or 150 seconds at a concentration of 0.1 μg/ml (antibodies 19H6-F7,3A4-H4, 54C1-H11 and 37D3-H9) using a flow rate of 10 μl/min. Binding ofTau monomer in HBSP was monitored at 25° C. using a flow rate of 30μl/min and concentrations of 16, 31, 63, 125, 125, 250 and 500 nM forantibodies 26C1 and 94B2; 16, 31, 63, 125, 125, 250, 500 and 1000 nM forantibodies 52F6-F11.v1 and 52F6-F11.v2; 6, 19, 56, 56, 167 and 500 nMfor antibodies 11E10-B8, 55E7-F11 and 125B11-H3; 5, 16, 49, 148, 148,444, 1333 and 4000 nM for antibodies 123E9-A1, 30G1-B2, 66F5-A1,89F4-A1, 93A8-D2 and 126F11-G11; 0.4, 1.6, 6.3, 2.5, 100 and 400 nM for19H6-F7; and 0.2, 0.8, 4, 4, 20 and 100 nM for 3A4-H4, 54C1-H11 and37D3-H9. Association and dissociation times were monitored for 180-480seconds and for 300-600 seconds respectively. Antibody 37D3-H9 wasselected for further analysis due to the high affinity (Table 6) and theabsence of NXS/T glycosylation motifs in the CDRs.

TABLE 6 K_(D) (nM) of murine antibodies to human Tau monomer. Data shownrepresent output of a 1:1 binding model. Antibody K_(D) (nM) k_(on)(1/Ms) k_(off) (1/s) 26C1 17 4 × 10⁴ 7 × 10⁻⁴ 94B2-C1 6 5 × 10⁴ 3 × 10⁻⁴54C1-H11 0.6 3 × 10⁵ 2 × 10⁻⁴ 3A4-H4 12 3 × 10⁴ 3 × 10⁻⁴ 37D3-H9 1.6 1 ×10⁵ 1 × 10⁻⁴ 19H6-F7 10 2 × 10⁵ 2 × 10⁻³ 11E10-B8 108 2 × 10⁵ 2 × 10⁻²55E7-F11 171 2 × 10⁵ 4 × 10⁻² 125B11-H3 5 5 × 10⁴ 3 × 10⁻⁴ 123E9-A1 52 4× 10⁵ 2 × 10⁻² 30G1-B2 20 4 × 10⁵ 8 × 10⁻³ 66F5-A1 105 8 × 10⁴ 8 × 10⁻³89F4-A1 27 3 × 10⁵ 7 × 10⁻³ 93A8-D2 6 3 × 10⁵ 2 × 10⁻³ 126F11-G11 3 2 ×10⁶ 4 × 10⁻³ 52F6-F11.v1 15 5 × 10⁴ 7 × 10⁻⁴ 52F6-F11.v2 5 7 × 10⁴ 4 ×10⁻⁴37D3-H9 Demonstrates Avidity when Binding to Tau Protein

Human monomer Tau protein was covalently coupled to a Biacore Series SCM5 chip using the Biacore Amine Coupling Kit (GE Life Sciences),resulting in immobilization to a level of approximately 128 RU. Directbinding of 37D3-H9 in both Fab and IgG formats was monitored using thesingle-cycle kinetics experimental format with five association periodsof 300s each and antibody concentrations of 1, 2, 4, 8 and 16 nM (IgG)or 5, 10, 20, 40 and 80 nM (Fab). Dissociation was monitored for 7200seconds (Fab) or for 14400 seconds (IgG). A value for the dissociationrate was calculated by fitting a 1:1 binding model to the data.Calculated dissociation rates were 5.0×10⁻⁴ for 37D3-H9 Fab and 1.1×10⁻⁵for 37D3-H9 IgG, a 45-fold difference. FIG. 5 illustrates the differencein the dissociation rates of Fab (left panel) and IgG (right panel),indicating that 37D3-H9 IgG is demonstrating avidity.

Example 4: Humanization of Anti-Tau Antibodies

Antibody 37D3-H9 was humanized by grafting the antibody CDRs andselected variable region framework residues onto human antibodyconsensus frameworks (Dennis, M.S. (2010). CDR repair: A novel approachto antibody humanization. In Current Trends in Monoclonal AntibodyDevelopment and Manufacturing, S. J. Shire, W. Gombotz, K.Bechtold-Peters and J. Andya, eds. (Springer, New York), pp. 9-28).Grafting onto consensus VH3, Vκ2 and W1 frameworks was assessed. Theheavy chain graft included murine residue at position 49 (Kabatnumbering system). The Vκ2 graft included murine residues in frameworkpositions 2 and 4. The W1 graft included murine residues in frameworkpositions 2, 4 and 43. Humanized variants were constructed by genesynthesis and subcloning into human IgG1 or IgG4 and Kappa chainmammalian expression vectors. Antibodies were expressed byco-transfection of the heavy and light chain plasmids into CHO cells andpurified with affinity resin MabSelect Sure. Humanized variants werescreened for affinity to human Tau monomer using the Biacore human IgGcapture kit, a Series S CM5 chip and a Biacore T200 instrument.Antibodies were diluted to 2 μg/ml and captured for 15 seconds at 10μl/min. Association and dissociation of 100, 33, 11 and 3.7 nM human Taumonomer in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (runningbuffer, HBSP) was monitored for 180 seconds and 600 seconds respectivelyat a flow rate of 30 μl/min. A 1:1 binding model was applied to theresults (Table 7).

TABLE 7 Affinity screening of humanized variants for monomeric human TauLight chain Antibody variant framework K_(D) (nM) hu37D3-H9.v1 Kappa14.1 hu37D3-H9.v2 Kappa1 5.6 hu37D3-H9.v3 Kappa1 8.8 hu37D3-H9.v4 Kappa18.2 hu37D3-H9.v5 Kappa2 1.9 hu37D3-H9.v6 Kappa2 3.5 hu37D3-H9.v7 Kappa227.0 hu37D3-H9.v8 Kappa2 10.2 hu37D3-H9.v9 Kappa2 13.2 hu37D3-H9.v10Kappa2 14.3 hu37D3-H9.v11 Kappa2 74.8 hu37D3-H9.v12 Kappa2 21.6hu37D3-H9.v13 Kappa2 9.0 hu37D3-H9.v14 Kappa2 10.8 hu37D3-H9.v15 Kappa219.0 hu37D3-H9.v16 Kappa2 27.2 hu37D3-H9.v17 Kappa2 8.1 hu37D3-H9.v18Kappa2 13.4 hu37D3-H9.v19 Kappa2 55.7 hu37D3-H9.v20 Kappa2 36.9hu37D3-H9.v21 Kappa2 38.1 hu37D3-H9.v22 Kappa2 36.6 hu37D3-H9.v23 Kappa281.1 hu37D3-H9.v24 Kappa2 56.6

Antibody variants hu37D3-H9.v1, hu37D3-H9.v2, hu37D3-H9.v5 andhu37D3-H9.v6 were characterized further by surface plasmon resonancewith additional antibody concentrations and longerassociation/dissociation times. These variants were analyzed with abroader range of human Tau monomer concentrations (1.2, 3.7, 11.1, 11.1,33.3, 100 nM) and increased association (300 seconds) and dissociation(1200 seconds) periods. A 1:1 binding model was applied to the results(Table 8).

TABLE 8 Detailed analysis of binding kinetics of selected variants tohuman Tau by surface plasmon resonance Antibody Light chain variantframework K_(D) (nM) hu37D3-H9.v1 Kappa1 1.1 nM, 1.0 nM hu37D3-H9.v2Kappa1 1.2 nM hu37D3-H9.v5 Kappa2 0.8 nM hu37D3-H9.v6 Kappa2 1.4 nM

A YTE (M252Y/S254T/T256E) mutation was incorporated into certain IgG₄antibodies. Fc Receptor-neonate (FcRn) binding domain mutations such asM252Y, S254T and T256E (YTE) have been described to increase FcRnbinding and thus increase the half-life of antibodies. See U.S.Published Patent Application No. 2003/0190311 and Dall'Acqua et al., J.Biol. Chem. 281:23514-23524 (2006).

Antibody 125B11-H3 was humanized onto VH3 and Vκ 1 consensus frameworks.The heavy chain graft included murine residues at position 78 (Kabatnumbering system). The Vκ1 graft included murine residues in frameworkpositions 43 and 87. The light chain of 113F5-F7 was also humanized ontothe Vκ1 framework, with additional murine residues at frameworkpositions 43 and 87. Humanized variant heavy chains (125B11) and lightchains (125B11 and 113F5-F7) were co-transfected in multiplecombinations and purified in 96-well format as described above.Humanized variants were then screened for affinity for human Tau monomerusing the Biacore human IgG capture kit, a Series S CM5 chip and aBiacore T200 instrument. Antibodies were diluted to 2 μg/ml and capturedfor 15 seconds at 10 μl/min. Association and dissociation of 0, 100 and500 nM human Tau monomer in HBSP was monitored for 180s and 300srespectively at a flow rate of 40 μl/min. A 1:1 binding model wasapplied to the results (Table 9).

TABLE 9 Screening of 125B11-H3 and 113F5-F7 humanization variants bysurface plasmon resonance 125B11 heavy chain humanization variantScreening K_(D) (nM) HC1 HC2 HC3 HC4 HC5 HC6 125B11 light LC1 16, 19 1818 15  85 — chain LC2 20 20 19 14  —* NT humanization LC3 21 23 20 15 —— variant LC4 23 22 20 17 >100 >100 113F5-F7 LC1 57 61 54 44 — — lightchain LC2 67 68 55 47 — — humanization LC3 61 64 54 47 >100 — variantLC4 71 77 65 51 — — *Minimal binding to Tau monomer. NT, not tested.

Variants hu125B11.v17 (HC3+LC1), hu125B11.v26 (HC4+LC2) and hu125B11.v28(HC4+LC4) were selected for high-resolution kinetic analysis based onthe affinity screen (Table 10). Antibody 94B2-C1 was humanized onto VH1and Vκ2 frameworks. The heavy chain graft also included murine residuesat position 28, 29, 67, 69, 71, and 73 (Kabat numbering system). The Vκ2graft also included murine residues in framework positions 2, 36, and46. Combinations of eight heavy chains and eight light chains wereexpressed, purified and screened by surface plasmon resonance (SPR) asdescribed for 125B11 above. Results of the SPR screen are shown in Table11. Variant hu94B2.v105 (heavy chain variant 94B2.HCl, light chainvariant 94B2.LC13) was selected for detailed SPR characterization (Table11).

TABLE 10 Kinetic data for selected humanized anti-Tau antibody variantsK_(D) K_(on) k_(off) Antibody Isotype (nM) (1/Ms) (1/s) hu125B11.v17hIgG1 10.5 0.8 × 10⁵ 0.8 × 10⁻³ hu125B11.v26 hIgG1 9.5 0.7 × 10⁵ 0.7 ×10⁻³ hu125B11.v28 hIgG1 10.2 0.7 × 10⁵ 0.7 × 10⁻³ hu94B2.v105 hIgG1 3.70.8 × 10⁵ 0.3 × 10⁻³

TABLE 11 Screening of 94B2 humanization variants by surface plasmonresonance 94B2 Light Chain humanization variant: Screening K_(D) (nM)LC9 LC10 LC11 LC12 LC13 LC14 LC15 LC16 94B2 HC1  3.8* ^(§) 91.5 ^(§)4.1^(¶) ^(§) 104.0  ^(§) Heavy HC2  5.7 ^(§) 89.6 ^(§) 7.4  NT 99.6 ^(§)Chain HC3  2.0 ^(§) 69.3 ^(§) 3.8  ^(§) 64.1 ^(§) human- HC4 61.9 ^(§)^(§) ^(§) 64.1  ^(§) ^(§) ^(§) ization HC5  2.7 ^(§) 62.6 ^(§) 4.0  ^(§)72.6 ^(§) variant: HC6  0.9 ^(§) 70.1 ^(§) 3.0  ^(§) 74.1 ^(§) HC7 52.9^(§) ^(§) ^(§) 57.8  ^(§) ^(§) ^(§) HC8  1.0 ^(§) 44.3 ^(§) 2.4  ^(§)51.5 ^(§) *Mean of n = 3 repeats. ^(¶)hu94B2.v105. ^(§) Minimal bindingto Tau monomer observed. NT, not tested.

Example 5: Stability Analysis of Humanized Anti-Tau AntibodiesIdentification of Chemical Instability

Antibody samples were thermally stressed to mimic stability over theshelf life of the product. Samples were buffer exchanged into 20 mMAcetate buffer, pH 5.5, or phosphate buffer, pH 7.4, and diluted to aconcentration of 1 mg/ml. One ml of sample was stressed at 40° C. for 2weeks and a second was stored at −70° C. as a control. Both samples werethen digested using trypsin to create peptides that could be analyzedusing liquid chromatography (LC)-mass spectrometry (MS) analysis. Foreach peptide in the sample retention time, from the LC as well as highresolution accurate mass and peptide ion fragmentation information(amino acid sequence information) were acquired in the MS. Extracted ionchromatograms (XIC) were taken for peptides of interest (native andmodified peptide ions) from the data sets at a window of ±10 ppm andpeaks were integrated to determine area. Relative percentages ofmodification were calculated for each sample by taking the (area of themodified peptide) divided by (area of the modified peptide plus the areaof the native peptide) multiplied by 100. These relative percentageswere then compared between the control (t=0) and the stressed (t=2weeks) samples. Percentages shown represent the control (t=0) valuesubtracted from the stressed (t=2 weeks) value. Deamidation analysis ofantibodies hu37D3-H9.v1 and hu37D3-H9.v5 led to the observation that thesequence N²⁸G²⁹N³⁰ (Kabat numbering) within the light chain CDR-1 wassusceptible to deamidation. The increase in deamidated N²⁸G²⁹N³⁰ wasfound to be 16.5% for hu37D3-H9.v1 and 11% for hu37D3-H9.v5.

Impact of Deamidation on Antibody Binding to Antigen

To assess the impact of N²⁸ deamidation on the affinity for human Tau,it was desirable to obtain two samples with widely separated N²⁸deamidation status. Hu37D3-H9.v5 hIgG4.S228P was incubated at 40° C. fortwo weeks at a concentration of 1 mg/ml in Phosphate Buffered Saline, pH7.4. Deamidation of the N²⁸G²⁹ motif was measured using LC-MS/MS. Thet=2 week stressed sample had a 43.1% increase deamidation relative tothe t=0 unstressed sample. The stressed and unstressed antibodies wereanalyzed for Tau binding by surface plasmon resonance (Biacore) usingthe GE Biacore human IgG capture kit and a Series S CM5 chip. The hIgGwere diluted to 2 μg/ml in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween20 (running buffer, HBSP) and captured at a flow rate of 10 μl/min for15 seconds (t0 sample) or 17 seconds (t2 sample). Kinetic data wascollected for Human Tau monomer injected at concentrations of 0, 3.1,6.3, 12.5, 25, 25, 50 & 100 nM in HBSP, using a flow rate of 30 μl/min,a 300 s association phase and an 1800 s dissociation phase. Betweencycles the surface was regenerated using a 30 second injection of 3MMagnesium Chloride at 10 μl/min. A 1:1 binding model was fitted to thedata using instrument defaults, including local fitting of the “RI”parameter. Results shown in FIG. 6 and Table 12 demonstrate thatalthough the stressed antibody immobilized at greater levels than theunstressed antibody in this experiment, the magnitude of the Tau bindingsignal (as represented by the magnitude of the parameter Rmax) wasnoticeably lower. After normalizing the Rmax value for the differencesin capture level, the stressed (t=2 weeks) sample appeared to showapproximately half the total Tau binding capacity of the unstressedsample (indicated by a 56% reduction in the Normalized Rmax). Thecalculated affinity did not appear to change: in this analysis thedifference in K_(D) between the t=0 and the t=2 weeks samples was lessthan 2% (K_(D)=0.7 nM for t=0 and t=2 weeks). The results are consistentwith the t=2 weeks sample containing a significantly reduced populationof high affinity antibody.

TABLE 12 Relative binding of stressed and unstressed hu37D3-H9.v5samples to monomeric Tau by surface plasmon resonance hu37D3-H9.v5Ligand Normalized Change in hIgG4.S228P Level Rmax Rmax (= Rmax/Normalized sample (RU) (RU) Ligand Level) Rmax Control (t = 0) 102.947.7 0.46 N/A Stressed (t = 2 weeks) 146.8 30.2 0.21 −56%

Impact of Deamidation on Antibody Binding to Antigen and Calculation of“Normalized Rmax”

Given that asparagine deamidation is expected to result in aspartic acidand iso-aspartic acid products (Bischoff R. & Kolbe H. V. J. (1994). J.Chromat. 5, 662, p 261-278) the impact of replacing N²⁸ with D²⁸(variant hu37D3-H9.v5 N28D) on affinity for human Tau monomer wasanalyzed. Affinity was assessed at 25° C. using a Biacore T200instrument, the GE Biacore human IgG capture kit and a CM5 Series Schip. The hIgG were diluted to 2 μg/ml in 10 mM HEPES pH7.4, 150 mMNaCl, 0.05% Tween 20 (running buffer, HBSP) and captured at a flow rateof 10 μl/min for 22 seconds. Kinetic data was collected for human Taumonomer injected at concentrations of 0, 6.3, 12.5, 25, 25, 50, 100,200, and 400 nM in HB SP, using a flow rate of 30 μl/min, a 300 secondassociation phase and a 600 second dissociation phase. Between cyclesthe surface was regenerated using a 30 second injection of 3M MagnesiumChloride at 10 μl/min. A 1:1 binding model was fitted to the data andaffinities for hu37D3-H9.v5 and hu37D3-H9.v5.3 (also referred to hereinas hu37D3-H9.v5 N28D) calculated using kinetic analysis. Parameters usedfor the 1:1 fitting included the Instrument default of local fitting forthe “RI” parameter. The results are shown in FIG. 7 and Table 13.

Calculated K_(D) for the hu37D3-H9.v5 N28D variant was 160×10⁻⁹ M,compared to 1.5×10⁻⁹ M (mean, n=4 intra-experiment determinations) forhu37D3-H9.v5 analyzed under the same conditions. Therefore, conversionof N²⁸ to D²⁸ causes >100-fold reduction in affinity. Given thecomparatively low affinity of the hu37D3-H9.v5 N28D variant, and thecomparatively rapid kinetics, we reasoned that the kinetics analysis ofa mixture of the N²⁸ and D²⁸ variants would be dominated by the higheraffinity population, and that presence of the lower affinity variantsmight be reflected by a reduction in the Normalized Rmax. To validatethis reasoning, the Tau-binding profile of antibody variantshu37D3-H9.v5 and hu37D3-H9.v5 N28D were compared to that of the twoantibodies mixed together in equal quantities. Compared to hu37D3-H9.v5alone, a 1:1 mix of hu37D3-H9.v5 and hu37D3-H9.v5 N28D resulted in a 45%reduction in Normalized Rmax (Table 13). We concluded that changes inNormalized Rmax upon thermal stress may be indicative of a reducedpopulation of high affinity antibody in the stressed sample. We reasonedthat changes in Normalized Rmax could therefore be used to screenvariants of hu37D3-H9 for improved stability.

TABLE 13 Changes in Normalized Rmax observed upon thermal stress ofhu37D3-H9.v5 and upon mixing of hu37D3-H9.v5 with anticipateddeamidation product hu37D3-H9.v5 N28D Decrease in Normalized Rmax K_(D)Rmax compared to Sample (nM) (RU) Reference* Comments hu37D3-H9.v5 1.5 ±0.2 76.1 ± 0.4 Reference Mean +/− Standard hIgG1 Deviation of fourintra- experiment analyses hu37D3-H9.v5 N28D 160 81.0  4% hIgG1hu37D3-H9.v5 & 2.0 46.4 45% Two antibodies hu37D3-H9.v5 N28D mixed at a1:1 ratio hIgG1 hu37D3-H9.v5 1.5 68.8  3% Control for hIgG4.S228P, t = 0Stressed sample hu37D3-H9.v5 1.5 33.4 54% Stressed sample hIgG4.S228P, t= 2 weeks *Normalized Rmax = Rmax (RU)/Ligand Level (RU). NormalizedRmax for reference antibody = 0.33 (mean of four intra-experimentdeterminations, standard deviation <0.01).

Antibody Optimization and Selection

Ninety 37D3-H9 variants were assessed by Biacore to compare theirfunctional stability with or without a two-week 40° C. thermal stressperiod. The variants included most single mutations of the N²⁸G²⁹N³⁰T³¹motif, double mutants containing the G29A mutation, double mutations ofAsn-28 and Tyr-32 that might functionally replace these tohydrogen-bonded residues, as well as all possible permutations ofresidues 2, 4, 33, and 93 as either the residues present in the original37D3-H9 antibody or the corresponding germline residue variant. Inaddition, mutations were tested in the context of residue 1 being Asp orGlu, which does not impact affinity or stability of the Asn-28 residue.

Antibodies were expressed by transient transfection of Expi293 cells in96-well format and automated purification performed on a Tecan freedomEVO 200 liquid handling system with a 500 μL MCA96 head. Briefly, IgGsin 1 mL culuture were captured using tip columns that were custom packedwith 20 μL Mab Select SuRe resin (Glygen Corp & GE Healthcare). Afterwashing with 1×PBS pH 7.4, IgGs were eluted into 160 μL of 50 mMphosphoric acid pH 3 and neutralized with 12 μL of 20×PBS pH 11.MabSelect SuRe tip columns were stripped in 0.1 M NaOH and regeneratedwith 1×PBS pH 7.4 for consecutive use of up to 15 times. Purifiedantibodies in 96-well format were normalized to 0.1 mg/ml using aHamilton Star liquid handling robot. The “pre-stress” samples were keptat approximately 4° C. and the “post-stress” samples were incubated at40° C. for two weeks in a PCR machine. Functional stability of thevariants was compared by running surface plasmon resonance kineticsexperiments with the “pre-stress” and “post-stress” antibody preps. Theantibodies were assessed using a human antibody capture CM5 Series Schip generated using the GE Biacore human IgG capture kit and a BiacoreT200 instrument. Antibodies diluted to 2 μg/ml were immobilized using a15 second injection time and 10 μl/min flow rate. Binding to Tau monomerat 0 nM, 26.5 nM and 265 nM, at 25° C., using a flow rate of 40 μl/min,was monitored for a 180 second association phase followed by a 300second dissociation phase. Samples were run in 10 mM HEPES pH7.4, 150 mMNaCl, 0.05% Tween 20 (HBSP) using a multi-cycle kinetics format. Datawas analyzed using BIAevaluation software, fitting a 1:1 binding model.The resulting affinity (K_(D)) values are shown in FIG. 8A-D. AStability Index was also calculated, using the rationale thataffinity-compromised antibodies (due for example to deamidation of keyresidues) are expected to contribute equally to the IgG capture level(“Ligand Level”) but to contribute less to the measured Tau binding, andthat this would be reflected in the experimentally derived value forRmax. To account for variations in the amount of each antibody captured,Rmax was normalized for the antibody capture level (as measured by“Ligand Level”, Response Units immobilized during antibody capture).Thus Normalized Rmax is calculated as the experimental Rmax (units=RU)divided by the “Ligand Level” (Evaluation output representing the RUcaptured during the hIgG capture step, units=RU), and Stability Index iscalculated here as Normalized Rmax (post-stress) divided by NormalizedRmax (pre-stress).

Selected antibodies were expressed by transient transfection of CHOcells and purified. The antibodies were then stressed for two weeks at 1mg/ml and deamidation analyzed by LC-MS/MS, using RCM tryptic peptidemapping with DTT reduction, IAA capping and pH 8.2 digestion. Results(Table 14) demonstrated that variant hu37D3-H9.v28.A4 had reducedsusceptibility to deamidation on the N²⁸G²⁹N³⁰ motif. The reduceddeamidation of the hu37D3-H9.v28.A4 was unexpected, as the residue isnot located in the immediate vicinity of the Asn-28 residue (FIG. 9) andit is not clear how the F33L mutation might stabilize Asn-28.

TABLE 14 Stability of the hu37D3-H9.v28.A4 variants in stress tests fordeamidation Increase in deamidation of light Antibody Thermal StressConditions chain N²⁸G²⁹N³⁰ hu37D3-H9.v1 hIgG1 40° C. in Acetate Buffer,pH 5.5 16.5% hu37D3-H9.v5 hIgG1 40° C. in Acetate Buffer, pH 5.5 11%hu37D3- hIgG1 40° C. in Acetate Buffer, pH 5.5 N²⁸: 2.8% H9.v28.A4 N³⁰:0.2% 37° C. in PBS pH 7.4 N²⁸: 5.3% N³⁰: ND hIgG4.S228P.YTE 40° C. inAcetate Buffer, pH 5.5 N²⁸: 0% N³⁰: 0% 37° C. in PBS pH 7.4 N²⁸: 10.4%N³⁰: 2.0%

Example 6: Humanized Anti-Tau Antibody Selection and CharacterizationAntibody Selection and Characterization: Binding to Human Tau Protein

Affinity of selected antibodies was assessed at 25° C. using a BiacoreT200 instrument, the GE Biacore human IgG capture kit and a CM5 Series Schip. The hIgG were diluted to 0.25 μg/ml in 10 mM HEPES pH7.4, 150 mMNaCl, 0.05% Tween 20 (running buffer, HBSP) and captured at a flow rateof 10 μl/min for 150 seconds. Kinetic data was collected for Human Taumonomer injected at concentrations of 0, 0.4, 1.2, 3.7, 11, 11, 33 and100 nM in HBSP, using a flow rate of 30 μl/min, a 300 second associationphase and a 600 second dissociation phase. Between cycles the surfacewas regenerated using two sequential 30 second injections of 3M MgCl at10 μl/min. Data was fit to a 1:1 binding model (Table 15).

TABLE 15 Kinetic data for selected humanized anti-Tau antibody variantsK_(D) k_(on) k_(off) Antibody Isotype (nM) (1/Ms) (1/s) hu37D3- hIgG11.5 6.9 × 10⁵ 1.1 × 10⁻³ H9.v28.A4 hu37D3-H9.v5 hIgG1 1.0 7.5 × 10⁵ 0.8× 10⁻³ hu37D3-H9.v5 hIgG4.S228P 1.3 7.1 × 10⁵ 0.9 × 10⁻³ hu37D3-H9.v1hIgG4.S228P 2.0 6.7 × 10⁵ 1.3 × 10⁻³Antibody Characterization: Binding to Human Tau Protein inhIgG4.S228P.YTE Format

Affinity was assessed at 25° C. using a Biacore T200 instrument, the GEBiacore human FAb capture kit and a CM5 Series S chip. The hIgG werediluted to 0.5 μg/ml in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20(running buffer, HBSP) and captured at a flow rate of 10 μl/min for 180seconds. Kinetic data was collected for Human Tau monomer injected atconcentrations of 0, 0.4, 1.2, 3.7, 11, 11, 33 and 100 nM in HB SP,using a flow rate of 30 μl/min, a 300 second association phase and a 600second dissociation phase. Between cycles the surface was regeneratedusing two sequential 60 second injections of 10 mM Glycine pH 2.1. Datawas fit to a 1:1 binding model. Kinetic data are shown in Table 16.

TABLE 16 Binding kinetics of hu37D3-H9.v28.A4 hIgG4.S228P.YTE tomonomeric human Tau by surface plasmon resonance Antibody K_(D) k_(on)k_(off) Antibody preparation (nM) (1/Ms) (1/s) hu37D3-H9.v28.A4 Prep 11.4 6 × 10⁵ 9 × 10⁻⁴ hIgG4.S228P.YTE Prep 2 1.4 6 × 10⁵ 9 × 10⁻⁴

Antibody Characterization: Binding to Cynomolgus Monkey Tau Protein

Affinity was assessed at 25° C. using a Biacore T200 instrument, the GEBiacore human IgG capture kit and a CM5 Series S chip. The hIgG werediluted to 2 μg/ml in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20(running buffer, HBSP) and captured at a flow rate of 10 μl/min for 15seconds. Kinetic data was collected for Human Tau monomer injected at aminimum of five different non-zero concentrations between 1.2 and 100nM, with one replicate concentration. Kinetics were assessed using aflow rate of 30 μl/min, a 300 second association phase and a 600 seconddissociation phase. Between cycles a 30 second regeneration injection of3M Magnesium Chloride was performed at a flow rate of 10 μl/min. Theresults were fit to a 1:1 binding model. Kinetic data are shown in Table17.

TABLE 17 Affinity of humanized anti-Tau antibodies for monomericcynomolgus monkey Tau Ligand Level Rmax Antibody (RU) (RU) K_(D) (nM)k_(on) (1/Ms) k_(off) (1/s) hu37D3.v28.A4 113.9 62.6 0.7 17 × 10⁵ 1 ×10⁻³ hu37D3.v28.F1 126.9 61.2 1.3 12 × 10⁵ 2 × 10⁻³ hu37D3.v28.A12 162.685.2 1.0 17 × 10⁵ 2 × 10⁻³ hu37D3.v29.2 168.6 86.0 1.4 17 × 10⁵ 2 × 10⁻³hu37D3-H9.v5 125.1 55.5 0.6 15 × 10⁵ 1 × 10⁻³ hu37D3-H9.v1 130.2 51.70.8 20 × 10⁵ 1 × 10⁻³

Humanized antibodies hu37D3.v28.A4 and hu37D3.v28.F1 also bind tophosphorylated Tau (pTau).

Example 7: Pharmacokinetics of Anti-Tau Antibody

To evaluate the pharmacokinetics of the anti-Tau 37D3-H9 mIgG2a antibodyin vivo, C57BL/6 mice were administered a single intravenous (IV) orintraperitoneal (IP) bolus injection at a dose of 10 mg/kg to consciousmice. At various time points up to 28 days post-dose, plasma sampleswere collected to determined anti-Tau antibody concentrations.

The concentrations of the dosed antibody in mouse plasma were measuredwith a generic ELISA using a mouse anti-muIgG2a antibody coat, followedby adding plasma samples starting at a dilution of 1:100, and finishedby adding a mouse anti-muIgG2a-biotin conjugate, and then streptavidinconjugated to horseradish peroxidase for detection. The assay had astandard curve range of 1.56-200 ng/mL and a limit of detection of 0.16μg/mL. Results below this limit of detection were reported as less thanreportable (LTR).

FIG. 10 shows the results of the pharmacokinetic analysis for anti-Tau37D3-H9 mIgG2a. Anti-Tau 37D3-H9 mIgG2a had similar exposure andclearance in wild-type C57BL/6 mice as isotype control antibodies, witha clearance of 6.31 mL/day/kg.

To evaluate the pharmacokinetics of anti-Tau 94B2-C1 mIgG2a and anti-tau125B11-H3 mIgG2a in vivo, a single IP bolus injection of antibody wasadministered at a dose of 10 mg/kg to conscious C57BL/6 mice. At varioustime points up to 28 days post-dose, plasma samples were collected todetermined anti-Tau antibody concentrations.

The concentrations of the dosed antibody in mouse plasma and wasmeasured with a generic ELISA using a mouse anti-muIgG2a antibody coat,followed by adding plasma samples starting at a dilution of 1:100, andfinished by adding a mouse anti-muIgG2a-biotin conjugate, and thenstreptavidin conjugated to horseradish peroxidase for detection. Theassay had a standard curve range of 0.78-100 ng/mL and a limit ofdetection of 0.078 μg/mL. The concentrations were also measured with aspecific ELISA using recombinant Tau as the coat, followed by addingplasma samples starting at a dilution of 1:10, and finished by addinggoat anti-mIgG2a conjugated to horseradish peroxidase for detection. Theassay had a standard curve range of 0.078-10 ng/mL and a limit ofdetection of 0.0008 μg/mL. Results below this limit of detection werereported as less than reportable (LTR).

The results of those experiments are shown in FIGS. 16 and 17. Anti-Tau94B2 mIgG2a had similar exposure and clearance in wild-type C57BL/6 miceas an isotype control antibody when concentrations were analyzed using ageneric assay, but lower exposure and faster clearance whenconcentrations were analyzed using a specific assay. See FIG. 16. Theclearance determined by the generic assay was 4.06 mL/day/kg and thatdetermined by the specific assay was 7.53 mL/day/kg. These resultssuggest that the antibody may undergo in vivo changes over time thatcompromise its ability to recognize its target. Anti-Tau 125B11-H3mIgG2a had similar exposure and clearance in wild-type C57BL/6 mice asan isotype control antibody, regardless of which assay generated theconcentrations. See FIG. 17. The clearance determined by the genericassay is 4.96 mL/day/kg and that determined by the specific assay is4.90 mL/day/kg.

Table 18 shows the pharmacokinetic parameters for anti-Tau antibodies37D3-H9, 94B2-C1. and 125B11-H3 in mice.

TABLE 18 Pharmacokinetic parameters for anti-Tau antibodies Admin-istration Cmax AUCinf CL or CL/F Route Assay (μg/mL) (μg/mL*day)(mL/day/kg) 37D3- IV Generic 185 1590 6.31 H9 IP Generic 107 1680 6.7694B2- IP Generic 151 2460 4.06 C1 IP Specific 141 1330 4.91 125B11- IPGeneric 127 2020 4.96 H3 IP Specific 151 2040 4.90

To evaluate the pharmacokinetics of hu37D3.v28.A4 hIgG4.S228P andhu37D3.v28.A4 hIgG4-S228P.YTE antibodies in vivo, cynomolgus monkeys(Macaca fascicularis) were administered a single IV bolus injection at adose of 1 mg/kg to conscious moneys. At various time points up to 49days post-dose, plasma samples were collected to determined anti-Tauantibody concentrations.

The concentrations of the dosed antibody in monkey plasma and wasmeasured with a generic ELISA using a sheep anti-human IgG antibodycoat, followed by adding plasma samples starting at a dilution of 1:100,and finished by adding goat anti-human IgG conjugated to horseradishperoxidase for detection. The assay had a standard curve range of0.156-20 ng/mL and a limit of detection of 0.02 μg/mL. Results belowthis limit of detection were reported as less than reportable (LTR).

FIG. 11 shows the results of the pharmacokinetic analysis forhu37D3.v28.A4 hIgG4.S228P and hu37D3.v28.A4 hIgG4-S228P.YTE. In FIG. 11,each set of datapoints represents one animal and the lines represent theaverage for all animals in the antibody and assay group. Table 19 showsthe pharmacokinetic parameters for hu37D3.v28.A4 hIgG4.S228P andhu37D3.v28.A4 hIgG4-S228P.YTE in cynomolgus monkeys.

TABLE 19 Pharmacokinetic parameters for hu37D3.v28.A4 hIgG4.S228P andhu37D3.v28.A4 hIgG4-S228P.YTE in cynomolgus monkeys Cmax AUCinf CL VssAntibody Assay (μg/mL) (day*μg/mL) (mL/day/kg) (mL/kg) anti-gD hlgG4Generic 34.6 386 2.66 55.5 hu37D3.v28.A4 Generic 35.7 ± 2.59 559 ± 209 1.97 ± 0.743 71.9 ± 16.0 hIgG4.S228P Specific 35.4 ± 1.37 419 ± 89.92.47 ± 0.581 60.8 ± 3.49 hu37D3.v28.A4 Generic 34.5 ± 5.23 578 ± 43.51.74 ± 0.125 60.5 ± 1.87 hIgG4.S228P.YTE Specific 33.5 ± 2.72 520 ± 39.01.93 ± 0.139 56.5 ± 4.90

Example 8: Further Epitope Characterization of Anti-Tau Antibody

Following a comparison of 37D3-H9 binding to biotinylated Tau monomerand biotinylated peptide (MAPT_10-24), binding of 37D3-H9 to additionalbiotinylated peptides was also assessed. Nunc maxisorp 96-wellmicroplates were coated at 4° C. for >12 hours with Neutravidin dilutedto 2 μg/ml in 50 mM Sodium Carbonate Buffer, pH 9.6. All subsequentincubations were performed at room temperature. After coating, plateswere blocked with Superblock™ (PBS) Blocking Buffer (Thermo FisherScientific) for two hours then washed thoroughly with PBS, 0.05%Polysorbate 20. Wells were then exposed to biotinylated Tau peptides(Table 20) or Avi-tag biotinylated Tau monomer at 1 μg/ml for one hourand washed as previously. Peptides were synthesized using standardsolid-phase Fmoc chemistry (see, e.g., Fmoc solid phase peptidesynthesis: A practical approach; Chan, W. C., White, P. D., Eds.; OxfordUniversity Press: New York, 2000). Antibodies 37D3-H9 mIgG2a andhu37D3-H9.v5 hIgG1, serially diluted from 500 nM to 50 pM in 90%Superblock™ (PBS) Blocking Buffer, were allowed to bind biotinylated-Taucoated wells for 90 minutes. Wells were washed as previously and boundantibody detected with peroxidase-conjugated secondary antibody(Invitrogen/Life Technologies) diluted 1/1000 in Superblock™ BlockingBuffer (Rabbit anti-Mouse IgG or Goat Anti-Human IgG (H+L)respectively). After twenty minutes wells were washed as previously andsignal developed with TMB Microwell 2-Component Substrate (KPL).Reactions were stopped by addition of 1M Phosphoric Acid and absorbanceat 450 nm was measured with a SpectraMax M2 platereader.

TABLE 20 Peptide Sequences MAP T SEQ ID sequence Peptide sequence NO:MAPT(10-24) VMEDHAGTYGLGDRK 592 MAPT(2-24) AEPRQEFEVMEDHAGTYGLGDRK 593MAPT(2-34) AEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD 594 MAPT(10-44)VMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAG 595 LK

The results of that experiment are shown in FIG. 12. FIG. 12A showsbinding of each of the indicated antibodies for the indicated peptides.Antibodies 37D3-H9 and 94B2-C1 both showed strong binding to fragment10-24 in that experiment, and antibody 94B2-C1 also showed strongbinding to fragment 1-15. Antibodies 19F8-C11 and 123E9-A1 showed strongbinding to fragment 19-33, while antibody 89F4-A1 showed strong bindingto fragments 28-42 and 37-51. See FIG. 12A. Antibodies 37D3-H9 mIgG2aand hu37D3-H9.v5 hIgG1 both showed strong binding to Tau fragments 2-24and 2-34 and weaker binding to fragment 10-24. See FIGS. 12B and 12C.These results suggest that antibodies 37D3-H9 mIgG2a and hu37D3-H9.v5hIgG1 bind an epitope of Tau within amino acids 2-24 of the matureprotein.

In an alanine scanning substitution experiment, mutations Y18A and L20Awere found to abrogate binding by murine antibody 37D3-H9 to a Taufragment (fragment 2-21), suggesting that the antibody contacts theseTau residues. Using a series of 15mer offset peptides, it was found thatmurine antibody 37D3-H9 showed similar binding to fragment 9-23 as tofragment 10-24, and also showed moderate binding to fragments 7-21,8-22, and 11-25.

Example 9: Cell-Based Characterization of 37D3-119 Humanized AntibodiesMethods Primary Hippocampal and Microglial Culture andHippocampal-Microglial Co-Culture

Dissociated primary hippocampal neurons were prepared from embryonic day16-17 wild-type C57BL/6N mice. Cells were plated onto PDL/laminin-coated8-well chamber slides (Biocoat, 354688 Corning) at 25,000 cells/well.Cells were plated and maintained in NbActiv4 (BrainBits) and half of themedia was replaced twice a week. Recombinant tau and antibodies wereapplied to the culture at 18 cell divisions.

For microglial culture, cortices and hippocampi from postnatal day 1-2C57BL/6N mice were dissociated and grown in 10% FBS in DMEM in 225 mm²culture flasks for 10-12 days. The culture flasks were gently shaken todissociate microglia and the cells in 10% FBS in DMEM were replated ontoeither PDL/laminin-coated 8-well chamber slides at 30,000 cells/well forimaging or uncoated 48-well plates (3548, Corning) at 100,000 cells/wellfor cytokine assay. 4-5 hours after plating, cells were switched toserum-free low-glucose DMEM and maintained overnight before treatmentwith recombinant tau and antibodies.

Hippocampal-microglial co-cultures were prepared by replating microgliadissociated from 225 mm² culture flasks onto 18 DIV primary hippocampalneurons in 8-well slide chambers (12,500 microglia and 25,000 neuronsper one well). Co-cultures were treated with recombinant tau andantibodies 4 hours after microglia plating.

In Vitro Treatment of Recombinant Tau and Antibodies

For 18 DIV hippocampal cultures or hippocampal-microglial co-cultures,recombinant human oligomeric tau and antibodies (500 nM each at 1:1ratio) or controls were pre-incubated in neuron culture medium(conditioned medium from 18 DIV hippocampal culture:fresh NbActiv4 at1:1) for 1 hour at 37° C. before they were added to the cells. Cellswere incubated with the tau-antibody mix or control in the media for 72hours (hippocampal culture) or 48 hours (hippocampal-microglialco-culture). Cells were washed with PBS three times before fixation.

For microglia culture, recombinant human oligomeric tau and antibodiesor controls were pre-incubated at 125 nM each(immunocytochemistry/imaging) or 250 nM each (cytokine assay) inlow-glucose DMEM in the absence of serum for 1 hour at 37° C. prior tothe addition to the cells. For immunocytochemistry/imaging, cells wereincubated with the tau-antibody mix or controls for 10 minutes andwashed three times with PBS before fixation. For cytokine assay, cellswere incubated with the tau-antibody mix or control for 24 hours andmedium of each well was collected for cytokine assay.

Immunocytochemistry, Imaging, and Quanitificaton

Cells were fixed with 4% paraformaldehyde in PBS for 15 min andpermeabilized with 0.1% Triton X-100 in PBS for 10 minutes. 10% donkeyserum was used for blocking and cells were incubated with primaryantibodies in PBS overnight at 4° C., followed by incubation withAlexa-fluorophore-labeled secondary antibodies against appropriatespecies developed in donkey (Invitrogen). Primary antibodies used wereanti-tau (DAKO), rabbit anti-human tau developed against the human tauN-terminal region spanning amino acids 11-24, anti-MAP2 (ab5392, Abcam),and anti-Iba-1 (ab5076, Abcam). The slides were mounted with ProlongGold DAPI (P36935, Invitrogen) and no.1 coverslips.

Confocal fluorescent imaging was performed with a LSM780 (Carl Zeiss,Inc.) using Zen 2010 software (Carl Zeiss, Inc.). For imaging ofhippocampal cultures and hippocampal-microglial co-cultures, 5 z-stackimages at 0.98 μm intervals were collected using Plan Apochromat 20x/0.8M27 objective lens. For the MAP2 fragmentation assay, a maximumintensity z projection was created for the image stack and analyzedusing Metamorph (Molecular Devices, Sunnyvale, Calif.). A median filterand nearest neighbor deconvolution were used for noise reduction.Neurite and cell body lengths were analyzed using the neurite outgrowthmodule followed by morphological processing. Fragments less than 15pixels (6.225 μm) were normalized to total signal length to obtain ameasure of MAP2 fragmentation.

Microglia were imaged with a-Plan Apochromat 100x/1.46 M27 objective.Quantification of recombinant tau uptake in the cells was performed withImage J (1.43u, 64-bit, National Institute of Health). ROIs of cell areawere drawn manually using Iba-1 signal as a reference. Area andintegrated intensity of tau immunoreactivity of ROI were measured toobtain tau immunoreactivity normalized to area. All analyses wereperformed blinded to experimental conditions.

Results

The results of the experiment are shown in FIG. 13. As shown in FIG.13A, antibodies with full effector function were not protective againstTau toxicity in the neuron-microglia co-cultures. FIG. 13B shows imagesof neuron-microglia co-cultures contacted with oligomeric Tau andantibodies (bottom panels). Antibody 37D3-H9 hIgG4 and hu37D3-H9 hIgG1(N297G), which lack effector function, were protective against Tautoxicity, while 37D3-H9 hIgG1 was not.

Example 10: Dose-Dependent Reduction of Tau Pathology in Tau Tg MiceAdministered 37D3-119 IgG2a or 37D3-H9 IgG2a DANG

Transgenic mice expressing human Tau P301L under the Thy1 promoter (TauP301L-Tg) were maintained on a C57BL/6N (Charles River) background. TauP301L-Tg and wild type littermate mice were assigned to treatment groupsand dosed once weekly intraperitoneally (i.p.) with either IgG2a-control(anti-gp120) at 30 mg/kg, anti-tau 37D3-H9 WT IgG2a at 3, 10 or 30mg/kg, anti-tau 37D3-H9 DANG IgG2a at 3, 10 or 30 mg/kg. DANG refers toD265A/N297G mutations in IgG2a, which abrogate effector function. Allantibody-dosing solutions were prepared in 10 mM histidine pH 5.8, 6%sucrose, 0.02% Tween 20 at a concentration of 10 mg/ml. Treatmentstarted at 13 weeks of age. The mouse groups in the in vivo study weremales and staggered into 3 cohorts. In addition, 3 TauP301L-Tg mice wereharvested at age 3 months without undergoing any treatment in order todetermine the baseline level of pathology at the time of treatmentinitiation.

To harvest tissue, mice were anesthetized with 2.5% tribromoethanol(0.5m1 per 25 g body weight) and transcardially perfused with PBS.Brains were harvested and bisected. Right hemispheres were fixed in 4%paraformaldehyde overnight at 4° C. then transferred to phosphatebuffered saline prior to processing for immunohistochemistry. Lefthemispheres were sub-dissected on ice then frozen at −80° C. forbiochemical analysis. Tail clips were taken from all mice to confirmgenotypes.

Hemibrains were multiply embedded into a gelatin matrix usingMultiBrain® blocks (Neuroscience Associates, Knoxville, Tenn.) andsectioned coronally at 25 μm thickness. Within each block, the brainposition was randomized relative to genotype and treatment.Free-floating sections of individual mouse hemibrains or of MultiBrain®blocks were stained as previously described (Le Pichon et al., 2013,PLoS One, 8(4): e62342), but with washes in PBS instead of Iris bufferedsaline and primary antibody incubations at 4° C. instead of roomtemperature. Primary antibody was rabbit anti-pTau212/214 (generatedin-house; 0.01 μg/ml). To avoid high background staining, in the case ofmouse primary antibodies that were subtype specific, we used thecorresponding subtype-specific secondary antibody (eg. Biotinylatedanti-mouse IgG₃, Bethyl A90-111B).

Immunohistochemically stained slides were imaged using the Leica SCN400(Leica Microsystems; Buffalo Grove, Ill.) whole slide scanning system at200× magnification with a resolution of 0.5 μm/pixel, Regions ofinterest (ROIs) were manually drawn on 4 matched hippocampal levels peranimal, and the amount of staining in these ROIs was quantified in anautomated fashion using the two endpoints described below. All imageanalysis was performed blind to genotype and treatment groups. Forpositive pixel area analysis for quantitation of RIC stains, digitalimages of antibody-labeled brain sections were analyzed as previouslydescribed (Le Pichon et al., 2013). The percent area stained wascalculated by normalizing the total positive pixels to the total pixelarea of the ROI. The integrated intensity was calculated using theBeer-Lambert law, absorbance=− log(transinitted light intensity/incidentlight intensity), for the positive pixel areas only.

The results of that experiment are shown in FIG. 14. Administration ofanti-tau 37D3-H9 WT IgG₂a or anti-tau 37D3-H9 DANG IgG₂a resulted in adose-dependent reduction of pTau212/214 in the hippocampus.

Example 11: Humanized 37D3-H9 Kappa 1 Variants

Humanized antibody variants based on hu37D3-H9.v1, which has a kappa 1light chain, were made and tested for N²⁸ stability. An alignment of thelight chain variable region of the three variants tested withhu37D3-H9.v1 is shown in FIG. 18. The three variants differ from eachother in the light chain variable region: hu37D3.v39 contains themutation F33L, hu37D3.v40 contains the mutation G29T and hu37D3.v41contains the mutation N30Q.

Antibody samples were thermally stressed, as follows. Samples werebuffer exchanged into 20 mM histidine acetate, 240 mM sucrose, pH 5.5and diluted to a concentration of 1 mg/ml. One ml of sample was stressedat 40° C. for 2 weeks and a second was stored at −70° C. as a control.Both samples were then digested using trypsin to create peptides thatcould be analyzed using liquid chromatography (LC)-mass spectrometry(MS) analysis. For each peptide in the sample retention time, from theLC as well as high resolution accurate mass and peptide ionfragmentation information (amino acid sequence information) wereacquired in the MS. Extracted ion chromatograms (XIC) were taken forpeptides of interest (native and modified peptide ions) from the datasets at a window of ±10 ppm and peaks were integrated to determine area.Relative percentages of modification were calculated for each sample bytaking the (area of the modified peptide) divided by (area of themodified peptide plus the area of the native peptide) multiplied by 100.These relative percentages were then compared between the control (t=0)and the stressed (t=2 weeks) samples. Percentages shown represent thecontrol (t=0) value subtracted from the stressed (t=2 weeks) value. Theresults are shown in Table 21. The results demonstrate that the F33Lmutation is effective for reducing deamidation in a kappa 1 humanizedlight chains.

TABLE 21 Stability of the hu37D3-H9.v1 variants in stress tests fordeamidation. Increase in deamidation of Antibody light chain N²⁸G²⁹N³⁰hu37D3.v39 hIgG4.S228P.YTE N²⁸: 2.7% N³⁰: No significant increasedetected hu37D3.v40 hIgG4.S228P.YTE N²⁸: 12.1% N³⁰: 3.9% hu37D3.v41hIgG4.S228P.YTE N²⁸: 6.0% N³⁰: Residue replaced with glutamine

Affinity of the humanized antibody variants was measured at 25° C. usinga Biacore T200 instrument, the GE Biacore human FAb capture kit, and aCM5 Series S chip. Antibodies were diluted to 1 μg/ml in HBSP (10 mMHEPES pH7.4, 150 mM NaCl, 0.05% Tween 20) and captured at a flow rate of10 μl/min for 180 seconds. Kinetic data were collected for human Taumonomer injected at 1.2, 3.7, 11, 33 and 100 nM in HB SP using theSingle Cycle Kinetics methodology and a flow rate of 30 μl/min. Eachconcentration of Tau monomer was injected for a period of 3 minutes anddissociation was monitored for ten minutes. Between cycles, the surfacewas regenerated with two sequential one-minute injections of 10 mMglycine pH2.1. Data was fit to a 1:1 binding model using BIAEvaluationsoftware. Each antibody was analyzed twice within the experiment; datain Table 22 are shown as mean±range.

TABLE 22 Affinities of hu37D3-H9.v1 variants for monomeric Tau K_(D)(nM) k_(on) (1/Ms) K_(off) (1/s) hu37D3.v1 hIgG1 2.3 ± 0.3 6 ± 0.5 × 10⁵ 1 ± 0.1 × 10⁻³ hu37D3.v1 hIgG4 2.3 ± 0.3 6 ± 0.2 × 10⁵  1 ± 0.1 × 10⁻³hu37D3.v39 hIgG4.YTE 1.9 ± 0.2 6 ± 0.6 × 10⁵ 1 ± 0.02 × 10⁻³ hu37D3.v40hIgG4.YTE 4.4 ± 0.5 8 ± 0.9 × 10⁵ 3 ± 0.02 × 10⁻³ hu37D3.v41 hIgG4.YTE5.4 ± 0.3 9 ± 1.2 × 10⁵  5 ± 0.3 × 10⁻³

Example 12: Pharmacokinetics and Pharmacodynamics of hu37D3.v28.A4hIgG4-S228P and hu37D3.v28.A4 hIgG4-S228P.YTE in Cynomolgus Monkeys

To evaluate the pharmacokinetics and pharmacodynamics of hu37D3.v28.A4hIgG4.S228P and hu37D3.v28.A4 hIgG4-S228P.YTE antibodies in vivo, fiveconscious cynomolgus monkeys (Macaca fascicularis) per group wereadministered a single IV bolus injection at a dose of 50 mg/kg in thefirst phase. Anti-gD hIgG4 was used as a control, also at a dose of 50mg/kg. At various time points up to 35 days post-dose, plasma and CSFsamples were collected to determine anti-Tau antibody concentrations.After the final sample collection, the animals were allowed to recoverfor 63-64 days before initiation of the second phase. In the secondphase, the 15 animals from the first phase, plus 3 additional animals,were divided into two groups; the first group (n=9) was administeredantibody hu37D3.v28.A4 hIgG4.S228P and the second group (n=9) wasadministered hu37D3.v28.A4 hIgG4-S228P.YTE antibody, both at 50 mg/kg.Brains of 4 or 5 animals per group were harvested at 2 days and 10 dayspost-dose.

Human IgG₄ antibodies in cynomolgus monkey plasma, CSF, and brainhomogenate (described below) were measured with an ELISA using a sheepanti-human IgG monkey adsorbed antibody coat, followed by adding plasmasamples starting at a dilution of 1:100, CSF samples starting at adilution of 1:20, or brain homogenate samples starting at a dilution of1:10, and finished by adding a goat anti-human IgG antibody conjugatedto horseradish peroxidase monkey adsorbed for detection. Color wasdeveloped using 3,3′,5,5′-tetramethylbenzidine and neutralized using 1Mphosphoric acid. Samples were read at 450/620 nm. The assay has astandard curve range of 0.156-20 ng/mL and a limit of detection of 0.02ug/mL for plasma, 0.003 μg/mL for CSF, and 0.002 μg/mL for brainhomogenate. Results below this concentration were reported as less thanreportable (LTR).

The results of the pharmacokinetic analysis are shown in FIG. 19A(plasma) and 19B (CSF), and in Tables 23 and 24. Animals that weresuspected of being anti-therapeutic antibody positive (ATA+) wereexcluded from the analysis. These data show that introducing the YTEmutations in the Fc region of hu37D3.v28.A4 hIgG4.S228P slowed theperipheral and CSF clearance rates of the antibody by about two fold.

TABLE 23 Mean (± SD) plasma clearance and C_(max) estimates followingsingle IV bolus dose Plasma clearance C_(max) Antibody (mL/day/kg)(μg/mL) anti-gD hIgG4 1.67 ± 0.415 1950 ± 174 hu37D3.v28.A4 hIgG4.S228P2.09 ± 0.229 1970 ± 144 hu37D3.v28.A4 hIgG4-S228P.YTE 1.12 ± 0.233 1850± 156

TABLE 24 Mean (± SD) CSF C_(max) estimates following single IV bolusdose Antibody C_(max) (μg/mL) anti-gD hIgG4  1.39 ± 0.751 hu37D3.v28.A4hIgG4.S228P 0.910 ± 0.552 hu37D3.v28.A4 hIgG4-S228P.YTE  2.51 ± 1.93 

The brain concentration of the antibodies at 2 and 10 dayspost-injection was determined as follows. Brain tissue was weighed andthen homogenized in 1% NP-40 in phosphate-buffered saline containingcOmplete™, Mini, EDTA-free protease inhibitor cocktail tablets. Thehomogenized brain samples were then rotated at 4° C. for 1 hour beforespinning at 14,000 rpm for 20 minutes. The supernatant was isolated forbrain antibody measurement by ELISA, as described above. The results ofthat experiment are shown in FIGS. 21A-D. The concentration of antibodyhu37D3.v28.A4 hIgG4-S228P.YTE in the brain, and the ratio ofbrain:plasma concentration for antibody hu37D3.v28.A4 hIgG4-S228P.YTEtrended higher than antibody hu37D3.v28.A4 hIgG4. S228P.

The pharmacodynamics response in plasma was also determined. Theconcentration of total Tau in K₂EDTA plasma was determined using anelectrochemiluminescence (ECL) immunoassay (Roche ProfessionalDiagnostics (RPD), Penzberg, Germany). The Elecsys® immunoassay isvalidated for the quantification of total Tau in human CSF, and becauseof the similarity between human and cynomolgus monkey Tau, wasconsidered acceptable for the measurement of cynomolgus monkey Tau inCSF and plasma. The assay captures and detects amino acids 159-224 ofhuman and cynomolgus monkey Tau, a region present in all known isoforms,independent of phosphorylation state. The lower detection limit (LDL) ofthe assay is 1.01 pg/mL. The assay is tolerant to 15.0 mg/mL ofhu37D3.v28.A4 hIgG4-S228P.YTE.

The results of the pharmacodynamic analysis are shown in FIG. 20. Therewere 3 animals per group after excluding animals suspected of beingATA+, and another animal that lacked baseline values. Surprisingly,within the first day of dosing, plasma Tau levels rise to a greaterdegree in the animals treated with the YTE variant versus the non-YTEvariant. Further, that result is not predicted from the pharmacokineticsresponse (FIG. 20), as the PK is similar between the variants at theearly time points. A more robust response is sustained in the animalstreated with the YTE variant for the entire duration of sampling.

Example 13: Pharmacokinetics and Pharmacodynamics of hu37D3.v28.A4hIgG4-S228P.YTE in Cynomolgus Monkey Brain

To assess antibody pharmacokinetics in brain, twelve consciouscynomolgus monkeys (Macaca fascicularis) per group were administered asingle IV bolus injection of hu37D3.v28.A4 hIgG4-S228P.YTE at a dose of50 mg/kg. Anti-gD hIgG4 was used as a control, also at a dose of 50mg/kg. At various time points up to 42 days post-dose, plasma sampleswere collected to determine anti-Tau antibody concentrations. Inaddition, at various time points up to 42 days, 2 monkeys weresacrificed and brain and CSF concentrations of antibody were determined.

Antibody concentrations were determined substantially as described inExample 12.

FIG. 22A-B show the concentration of antibody in cynomolgus monkey brainat various time points post-dose, plotted in logarithmic (A) and linear(B) scale. Table 25 shows the brain concentration parameters.

TABLE 25 Mean (± SD) brain PK parameter estimates following single IVbolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 0.175 ±0.02 4.26 ± 0.35 hu37D3.v28.A4 hIgG4-S228P.YTE 0.12. ± 0.03 3.88 ± 0.89

The hu37D3.v28.A4 hIgG4-S228P.YTE antibody showed increased brainconcentration at the terminal timepoint, compared to anti-gD.

The concentration of the antibodies in various regions of the brain,including hippocampus, cerebellum, and frontal cortex, was alsodetermined. FIG. 23A-C and Tables 26 to 28 show the results of thatanalysis.

TABLE 26 Mean hippocampus PK parameter estimates following single IVbolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 0.1593.95 hu37D3.v28.A4 hIgG4-S228P.YTE 0.087 2.87

TABLE 27 Mean cerebellum PK parameter estimates following single IVbolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 0.1964.30 hu37D3.v28.A4 hIgG4-S228P.YTE 0.139 4.56

TABLE 28 Mean frontal cortex PK parameter estimates following single IVbolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 0.17 4.65hu37D3.v28.A4 hIgG4-S228P.YTE 0.138 4.22

The results of that experiment show exposure of various regions of thebrain to antibody hu37D3.v28.A4 hIgG4-S228P.YTE following a single IVinjection. Overall exposures in brain were comparable across the twogroups, however, similar to the observations in plasma, there was abouta two-fold increase in antibody concentrations in the brain at theterminal timepoint in animals dosed with antibody hu37D3.v28.A4hIgG4-S228P.YTE, compared to anti-gD. See FIG. 23. These results suggestmaintenance of higher trough (terminal) concentrations in brain afterdosing with the YTE antibody.

The concentration of the antibodies in CSF and plasma over time was alsodetermined. FIGS. 23D (CSF) and 23E (plasma) and Tables 29 and 30 showthe results of that analysis.

TABLE 29 Mean CSF PK parameter estimates following single IV bolus doseCmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 1.270 18.400hu37D3.v28.A4 hIgG4-S228P.YTE 3.980 21.100

TABLE 30 Mean plasma PK parameter estimates following single IV bolusdose Cmax Tmax AUCall Terminal Group (μg/ml) Day (day*μg/ml) (Day 43)anti-gD hIgG4 0.175 ± 0.02 2 4.26 ± 0.35 36.3 ± 14.1 hu37D3.v28.A4 0.12.± 0.03 3 3.88 ± 0.89 89.4 ± 42.3 hIgG4-S228P.YTE

Again, similar to the plasma and brain pharmacokinetics, there was abouta two-fold increased antibody concentration in CSF and plasma at theterminal timepoint in animals dosed with antibody hu37D3.v28.A4hIgG4-S228P.YTE, compared to anti-gD. See FIG. 23.

Using the collected plasma samples from the cynomolgus monkeys, theplasma pharmacodynamics of antibody hu37D3.v28.A4 hIgG4-S228P.YTE andcontrol antibody following a single IV 50 mg/kg dose were assessed.Plasma Tau was quantitated using the Elecsys® immunoassay discussed inExample 12.

The results of the pharmacodynamics analysis are shown in FIG. 24A-B.FIG. 24A shows the mean total plasma Tau concentration, normalized tobaseline. FIG. 24B shows the total plasma Tau concentration inindividual monkeys in the study, normalized to baseline. Similar to theresults observed in Example 12, administration of antibody hu37D3.v28.A4hIgG4-S228P.YTE resulted in significantly increased plasma Tau levels.While not intending to be bound by any particular theory, these datasuggest that hu37D3.v28.A4 hIgG4-S228P.YTE binds to Tau in the brain,and consequently Tau is cleared from the brain into the periphery. Theseresults are consistent with target engagement in the brain byhu37D3.v28.A4 hIgG4-S228P.YTE.

Example 14: Affinity Maturation of Antibody hu37D3.v28.A4

Affinity maturation of the 37D3.v28.A4 was done by deep sequencing of ascanning mutagenesis library. Two libraries were designed, one for eachantibody chain, in which selected positions in the heavy or light chainvariable regions were randomized with an NNK (IUPAC code) codon, whichencodes any amino acid or an amber stop codon. The design allows onlyone amino acid change in the antibody variable regions per clone. Thepositions were selected from CDRs and framework positions in directcontact or near to CDRs. Light chain Kabat positions 1 to 5, 24 to 27,29 to 36, 38, 43, 44, 46 to 58, 60, 63 to 71, 87 and 89 to 97 and heavychain Kabat positions 1, 2, 4, 24 to 39, 43, 45 to 81, 82a, 82b, 83, 85,86, 91 and 93 to 103 were randomized with NNK codons. The light chainclones that did not randomize position 28 had a Ser at position 28instead of the wild-type Asn. Two different DNA fragments were used torandomize position 28 of the light chain. One clone had a codon VNK andthe other an NYK codon, which allowed for any amino acid except Tyr,Cys, Trp and stop codons. The libraries were created by DNA synthesis(GeneWiz), producing 60 independent linear DNA fragments for the lightchain and 75 for the heavy chain, with one position in each fragmentrandomized with the NNK codon or the VNK/NYK mix. The linear DNAfragments were pooled for each chain and cloned into a monovalent Fabfragment phage display vector (see Lee C V et al., J. Immunol. Methods284:119-132 (2004)). The light chain library clones had a wild-typeheavy chain variable region whereas the heavy chain library clones had alight chain variable region with an N28S mutation. Ligation productswere electroporated into Escherichia coli XL-1, superinfected with M13KO7 helper phage (New England Biolabs) and grown as described (seeKoenig P et al., J. Biol. Chem. 290:21773-21786 (2015)).

Three sequential rounds of sorting were performed, using Tau monomerprotein adsorbed to an ELISA plate (Round 1) or a solution of anN-terminally biotinylated peptide encompassing residues 2 to 24(biotin-PEG-AEPRQEFEVMEDHAGTYGLGDRK; SEQ ID NO: 624) of human Tau(Rounds 2 and 3). In the first round, phage (1 OD₂₆₈/ml) were incubatedwith a Tau-coated ELISA plate for 2 hours at ambient temperature. In thesecond and third rounds, phage (5 OD₂₆₈/ml) were incubated with 100 nMbiotinylated Tau peptide for 2 hours at ambient temperature, thendiluted 5-fold and captured on ELISA plates coated with neutravidin(Pierce) for 10 minutes. Stringency was increased in Round 3 byincubating wells containing captured phage with binding buffer (PBS,0.5% Bovine Serum Albumin) for an additional 20 minutes after the phagecapture and washing. The wash step was then repeated. Phage were elutedfrom the ELISA plates by incubation with 0.1M hydrochloric acid and werepropagated by infection of Escherichia coli followed by superinfectionwith M13KO7 helper phage (New England Biolabs). To facilitate analysisby next-generation sequencing, in Round 3 a “mock” selection wasperformed in parallel with the selection just described. The “mock”selection, intended to provide a negative control or reference sample,was performed using the same method as the Round 3 just described butomitting the addition of biotinylated peptide.

Initial results were obtained by Sanger sequencing of individualcolonies.

In addition, plasmid DNA was extracted from the Escherichia coli XL-1populations used for phage amplification. Inserts were amplified by an18-cycle PCR amplification using Phusion DNA polymerase (New EnglandBiolabs) followed by agarose gel purification of amplicons. Ampliconswere sequenced by Illumina sequencing as previously described (KoenigP., et al. (2015). J. Biol. Chem. 290, 21773-21786). Sequences werefiltered using the PCR primer sequences and by removing sequences thatwere longer or shorter than the parental sequence, or contained morethan one coding mutation in the variable region. Enrichment ratios werecalculated by dividing the frequency of a given mutation at a givenposition in one population by the frequency of the very same mutation ina second population. Populations were named using the followingabbreviations:

R0—Unsorted library

R2—Phage from the Round 2 selection using 100 nM biotinylated peptide

R3—Phage from the Round 3 selection using 100 nM biotinylated peptide

R3M—Phage from the Round 3 “mock” selection.

Selected mutations were transferred, either individually or incombination, onto the hu37D3.v28.A4 variable domains, synthesized andcloned into an IgG mammalian cell expression vector. In some instances,selected mutations were transferred onto variable domains that that hadbeen humanized differently to hu37D3.v28.A4. Examples of alternativelyhumanized antibodies containing mutations identified by the phagedisplay procedures described above include hu37D3-H9.v76, hu37D3-H9.v83,and hu37D3-H9.v93.

Heavy chain and light chain plasmids were expressed in Expi293 cells bytransient transfection and IgG purified from the supernatants usingaffinity chromatography with resin Mab Select SuRe (GE Life Sciences).Purified IgG were analyzed for binding to human Tau monomer usingsurface plasmon resonance. Briefly, a Biacore T200 instrument was usedto capture antibodies on a human IgG capture chip that had beengenerated using a Series S CM5 Sensor Chip and the human IgG capture kit(GE Life Sciences). Captured antibodies were exposed to human Taumonomer in solution and the association and dissociation phasesmonitored. Affinities were calculated by fitting a 1:1 binding model tothe kinetic data using Biacore Evaluation software.

A comparison of the light and heavy chain variable regions forhu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93 versus the parenthu37D3-H9.v28.A4 is shown in FIGS. 25A-B. Black shading indicates aminoacids that are different between the affinity-matured and parentantibody.

The affinity-matured hu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93antibodies were evaluated for their monovalent interaction with humanand cynomolgus monkey recombinant Tau monomer.

Affinity for human and cynomolgus monkey Tau was measured at 37° C.using a Biacore T200 instrument, the GE Biacore human IgG capture kitand a Series S CM5 Sensor Chip. Antibodies were diluted to 1 μg/ml inrunning buffer HBS-EP (10 mM HEPES pH7.4, 150 mM NaCl, 1 mM EDTA, 0.05%Tween 20) and captured at a flow rate of 10 μl/min for 15 seconds. Datawas collected for the binding of human and cynomolgus Tau monomer, eachof which was injected at 0, 0.1, 0.4, 1.2, 3.7, 11.1, and 33.3 nM, usinga flow rate of 30 μl/min, a contact time of 300s, and a dissociationtime of 900s. Between cycles, the surface was regenerated with 3Mmagnesium chloride. Data was fit to a 1:1 binding model usingBIAEvaluation software.

FIG. 26A presents affinity data for the hu37D3-H9.v76 antibody for humanTau monomer, with each curve representing a different antibodyconcentration. FIG. 26B presents these data for cynomolgus monkey (cyno)Tau monomer. The affinity-purified hu37D3-H9.v76, hu37D3-H9.v83, andhu37D3-H9.v93 antibodies each had a K_(D) of 0.1 nM at 37° C. for bothhuman and cynomolgus monkey Tau monomers, as shown in Table 31. Inparallel experiments, the parent hu37D3-H9.v28.A4 antibody had a K_(D)of 5-8 nM (data not shown).

TABLE 31 Affinity measurement of hu37D3 affinity-matured antibodiesHuman Tau monomer Cyno Tau monomer KD (nM) ka (1/Ms) kd (1/s) KD (nM) ka(1/Ms) kd (1/s) hu37D3-H9.v76 0.1 6.94 × 10⁶ 8.92 × 10⁻⁴ 0.1 9.27 × 10⁶1.14 × 10⁻³ hu37D3-H9.v83 0.1 6.19 × 10⁶ 8.18 × 10⁻⁴ 0.1 9.55 × 10⁶ 1.14× 10⁻³ hu37D3-H9.v93 0.1 6.89 × 10⁶ 1.02 × 10⁻³ 0.1 1.29 × 10⁷ 1.78 ×10⁻³

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Table of Sequences SEQ ID NO Description Sequence 2 Human Tau epitopeAEPRQEFEVMEDHAGTYGLGDRK (2-24) 4 Cynomolgus AEPRQEFDVMEDHAGTYGLGDRKmonkey Tau epitope (2-24) 10 37D3-H9 heavyEVQLVESGGD LAKPGGSLKL SCTASGLIFR SYGMSWVRQT chain variablePDKRLEWVAT INSGGTYTYY PDSVKGRFTI SRDNAKNTLY region (VH)LQMSSLKSED TAMYYCANSY SGAMDYWGQG TSVTVSS 11 37D3-H9 light chainDDLLTQTPLS LPVSLGDPAS ISCRSSQSIV HSNGNTYFEW variable region (VL)YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKISRVEAEDLGV YYCFQGSLVP WTFGGGTKLE IK 12 37D3-H9 HVR-H1 SYGMS 1337D3-H9 HVR-H2 TINSGGTYTYYPDSVKG 14 37D3-H9 HVR-H3 SYSGAMDY 1537D3-H9 HVR-L1 RSSQSIVHSNGNTYFE 16 37D3-H9 HVR-L2 KVSNRFS 1737D3-H9 HVR-L3 FQGSLVPWT 20 37D3-H9b heavyEVQLVESGGD LAKPGGSLKL SCTASGLIFR SYGMSWVRQT chain variablePDKRLEWVAT INSGGTYTYY PDSVKGRFTI SRDNAKNTLY region (VH)LQMSSLKSED TAMYYCANSY SGAMDYWGQG TSVTVSS 21 37D3-H9b lightEDLLTQTPLS LPVSLGDPAS ISCRSSQSIV HSNGNTYFEW chain variableYLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDLGV YYCFQGSLVP WTFGGGTKLE IK 22 37D3-H9b HVR-H1 SYGMS 2337D3-H9b HVR-H2 TINSGGTYTYYPDSVKG 24 37D3-H9b HVR-H3 SYSGAMDY 2537D3-H9b HVR-L1 RSSQSIVHSNGNTYFE 26 37D3-H9b HVR-L2 KVSNRFS 2737D3-H9b HVR-L3 FQGSLVPWT 30 11E10-B8 heavyEVQLVESGGD LVKPGGSLKL SCAASGFTFR SYGMSWVRQT chain variablePDKRLEWVAT ISGGGSYTYY PDSVKGRFTI SRDNAKNTLY region (VH)LQMSSLKSED TAMYYCAVSY DGAMDYWGQG TSVTVSS 31 11E10-B8 lightDVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW chain variableYLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDLGL YYCFQGSHVP WTFGGGTKLE IK 32 11E10-B8 HVR-H1 SYGMS 3311E10-B8 HVR-H2 TISGGGSYTYYPDSVKG 34 11E10-B8 HVR-H3 SYDGAMDY 3511E10-B8 HVR-L1 RSSQSIVHSNGNTYLE 36 11E10-B8 HVR-L2 KVSNRFS 3711E10-B8 HVR-L3 FQGSHVPWT 40 54C1-H11 andEVQLVESGGD LVKPGGSLKV SCVASGFTFR SYGMSWVRQT 61E7-C4 heavyPDKRLDWVAT ISSGGNYTYY PDSVKGRFTI SRDNAKNTLY chain variableLQMSSLKSED TAMYYCASSY SGAMDYWGQG TSVTVSS region (VH) 41 54C1-H11 andDTVMTQSPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW 61E7-C4 light chainYLQKPGQSPK LLIYTVSNRF SGVPDRFSGS GSGTDFTLKI variable region (VL)SRVEAEDLGV YYCFQGSHVP WTFGGGTKLE IK 42 54C1-H11 and SYGMS 61E7-C4 HVR-H143 54C1-H11 and TISSGGNYTYYPDSVKG 61E7-C4 HVR-H2 44 54C1-H11 andSYSGAMDY 61E7-C4 HVR-H3 45 54C1-H11 and RSSQSIVHSNGNTYLE 61E7-C4 HVR-L146 54C1-H11 and TVSNRFS 61E7-C4 HVR-L2 47 54C1-H11 and FQGSHVPWT61E7-C4 HVR-L3 50 3A4-H4 heavyEVQLVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQT chain variablePDKRLEWVAT ISSGGTYTYY PDSVKGRFTI SRDNAKNTLY region (VH)LQMSSLKSED TAMYFCATSY DGAMDYWGQG TSVTVSS 51 3A4-H4 light chainDVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW variable region (VL)YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKISRVEAEDLGV YYCFQGTLVP WTFGGGTKLE IK 52 3A4-H4 HVR-H1 SYGMS 533A4-H4 HVR-H2 TISSGGTYTYYPDSVKG 54 3A4-H4 HVR-H3 SYDGAMDY 553A4-H4 HVR-L1 RSSQNIVHSNGNTYLE 56 3A4-H4 HVR-L2 KVSNRFS 57 3A4-H4 HVR-L3FQGTLVPWT 60 19H6-F7 heavy EVQLVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQTchain variable PDKRLEWVAT ISSGGTYTYY PDSVKGRFTI SRDNAKNTLY region (VH)LQMSSLKSED TAMYYCAPSY DGAMDYWGQG TSVTVSS 61 19H6-F7 light chainDVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW variable region (VL)YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKISRVEAEDLGV YYCFQGSLVP WTFGGGTKLE IK 62 19H6-F7 HVR-H1 SYGMS 6319H6-F7 HVR-H2 TISSGGTYTYYPDSVKG 64 19H6-F7 HVR-H3 SYDGAMDY 6519H6-F7 HVR-L1 RSSQSIVHSNGNTYLE 66 19H6-F7 HVR-L2 KVSNRFS 6719H6-F7 HVR-L3 FQGSLVPWT 70 94B2-C1 heavyEVQLQQSGPE LVKPGASMKI SCKASGYSLT GYTMNWVKQS chain variableHGKNLEWIGL ISPYNGVTSY NQKFKGKATL TVDKSSNTAY region (VH)MELLSLTFED SAVYYCARQG AYWGQGTLVT VSA 71 94B2-C1 light chainDVVMTQTPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW variable region (VL)LLQRPGQSPK RLIYLVSKLD SGVPDRFTGS GSGTDFTLKISRVEAEDLGV YYCWQGTHFP WTFGGGTKLE IK 72 94B2-C1 HVR-H1 GYTMN 7394B2-C1 HVR-H2 LISPYNGVTSYNQKFKG 74 94B2-C1 HVR-H3 QGAY 7594B2-C1 HVR-L1 KSSQSLLDSDGKTYLN 76 94B2-C1 HVR-L2 LVSKLDS 7794B2-C1 HVR-L3 WQGTHFPWT 80 125B11-H3 heavyEVKLEESGGG LVQPGGSMKL SCVASRFIFS NYWMNWVRQS chain variablePEKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKSS region (VH)VYLQMNNLRA EDTGIYYCTG GTTYWGQGTT LTVSS 81 125B11-H3 lightDIVMTQSQKF LSTSVGDRVN ITCKASQNVG TAVAWYQQKP chain variableGQSPGLLIYS ASIRYTGVPD RFTGNGSGTD FTLTISDMQS region (VL)EDLADYFCQQ FRTYPYTFGG GTKLEIK 82 125B11-H3 HVR- NYWMN H1 83125B11-H3 HVR- QIRLKSDNYA THYAESVKG H2 84 125B11-H3 HVR- GTTY H3 85125B11-H3 HVR- KASQNVGTAVA L1 86 125B11-H3 HVR- SASIRYT L2 87125B 1-H3 HVR- QQFRTYPYT L3 90 113F5-F7 heavyEVKLEESGGG LVQPGGSMRL SCVASEFTFS NYWMNWIRQS chain variablePEKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDASNFS region (VH)VYLQMNNLRA EDTGIYYCTG GTSYWGQGTT LTVSS 91 113F5-F7 lightDIVMTQSQKI MSTSVGDRVS ITCKASQNVG TAVAWYQQRP chain variableGHSPKLLIYS ASRRFSGVPD RFTGSGSGTD FTLTIINVQS region (VL)EDLADYFCQQ FSTYPYTFGV GTKLEIK 92 113F5-F7 HVR-H1 NYWMN 93113F5-F7 HVR-H2 QIRLKSDNYATHYAESVKG 94 113F5-F7 HVR-H3 GTSY 95113F5-F7 HVR-L1 KASQNVGTAVA 96 113F5-F7 HVR-L2 SASRRFS 97113F5-F7 HVR-L3 QQFSTYPYT 100 26C1-B11 heavyEVHLQQSGAE LVRSGASVKL SCTASGFNIK DYYMYWVKQR chain variablePEQGLEWIGW IDPENGDTEY FPKFQGKATM TADTSSKTAY region (VH)LQLSSLTSED TAVYYCNAWR ARATNSALDY WGQGTSVTVS S 101 26C1-B11 lightDVVMTQTPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW chain variableLLRRPGQSPK RLIYLVSKLD SGVPDRFTGS GSGTDFTLKI region (VL)SRVEAEDLGV YYCWQGTHFP WTFGGGTKLE IK 102 26C1-B11 HVR-H1 DYYMY 10326C1-B11 HVR-H2 WIDPENGDTE YFPKFQG 104 26C1-B11 HVR-H3 WRARATNSAL DY 10526C1-B11 HVR-L1 KSSQSLLDSD GKTYLN 106 26C1-B11 HVR-L2 LVSKLDS 10726C1-B11 HVR-L3 WQGTHFPWT 110 26C1-C8 heavyEVHLQQSGAE LVRSGASVKL SCTASGFNIK DYYMYWVKQR chain variablePEQGLEWIGW IDPENGDTEY FPKFQGKATM TADTSSKTAY region (VH)LQLSSLTSED TAVYYCNAWR ARATNSALDY WGQGTSVTVS S 111 26C1-C8 light chainDVVMTQTPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW variable region (VL)LLRRPGQSPK RLIYLVSKLD SGVPDRFTGS GSGTDFTLKISRVEAEDLGV YYCWQGTHFP WTFGGGTKLE IK 112 26C1-C8 HVR-H1 DYYMY 11326C1-C8 HVR-H2 WIDPENGDTE YFPKFQG 114 26C1-C8 HVR-H3 WRARATNSAL DY 11526C1-C8 HVR-L1 KSSQSLLDSD GKTYLN 116 26C1-C8 HVR-L2 LVSKLDS 11726C1-C8 HVR-L3 WQGTHFPWT 120 30G1-B2 heavyQVQLQQSGAE LVRPGASVTL SCKASGYTFT DYEMYWVKQT chain variablePVHGLEWIGA IDPETGDTAY NQKFKGKATL TADKSSNTAY region (VH)MELRSLTSED SAVYYCIRQY GNWFPYWGQG TLVTVSA 121 30G1-B2 light chainDVVMTQTPLS LPVSLGDQAS ISCRSSQSLV HANGNTYLHW variable region (VL)FLQKPGLSPK LLIYKVSNRF SGVPDRFSGG GSGTDFTLKITRLEAEDLGV YFCSQSTHVP FTFGSGTKLE IK 122 30G1-B2 HVR-H1 DYEMY 12330G1-B2 HVR-H2 AIDPETGDTAYNQKFKG 124 30G1-B2 HVR-H3 QYGNWFPY 12530G1-B2 HVR-L1 RSSQSLVHANGNTYLH 126 30G1-B2 HVR-L2 KVSNRFS 12730G1-B2 HVR-L3 SQSTHVPFT 130 66F5-A1 heavyQVQLQQSGAE LVRPGASVTL SCKASGYTFI DYEMNWVKQT chain variablePVHGLEWIGA IDPENGGTAY NQKFKGKAIV TADKSSSTAY region (VH)MELRSLTSED SAVYYCSGPH FDYWGQGTTL TVSS 131 66F5-A1 light chainDIVMTQSPSS LAMSVGQKVT MSCKSSQSLL NSSTQKNYLA variable region (VL)WYQQKPGQSP KLLVYFASTR ESGVPDRFIG SGSGTDFTLTISSVQAEDLA DYFCQQHYST PYTFGGGTKL EIK 132 66F5-A1 HVR-H1 DYEMN 13366F5-A1 HVR-H2 AIDPENGGTA YNQKFKG 134 66F5-A1 HVR-H3 PHFDY 13566F5-A1 HVR-L1 KSSQSLLNSS TQKNYLA 136 66F5-A1 HVR-L2 FASTRES 13766F5-A1 HVR-L3 QQHYSTPYT 140 123E9-A1 heavyEVQLQQSGPE LVKPGASVKM SCKASGYTFT DYYMKWVKQS chain variableHGKSLEWIGD IDPNNGGTSY NQKFKGKATL TVDKSSSTAY region (VH)MQLNSLTSED SAVYYCARSA GFGDSFSFWG LGTLVTVSA 141 123E9-A1 lightDVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW chain variableYLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDLGF YYCFQGSHVP PTFGGGTKLE IK 142 123E9-A1 HVR-H1 DYYMK 143123E9-A1 HVR-H2 DIDPNNGGTSYNQKFKG 144 123E9-A1 HVR-H3 SAGFGDSFSF 145123E9-A1 HVR-L1 RSSQSIVHSNGNTYLE 146 123E9-A1 HVR-L2 KVSNRFS 147123E9-A1 HVR-L3 FQGSHVPPT 150 15C6-A7 heavyEVQLQQSGPE LVKPGASVMM TCKASGYTFT DYYMKWVKQS chain variableNGKSLEWIGD LDPYTGGANY NQKFKGKATL TVDKSSSTAY region (VH)MHLNSLTSED SAVYYCARSR GYGDSFAYWG QGTLVTVSA 151 15C6-A7 light chainDVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW variable region (VL)YLQKPGQSPK LLIYKVSNRF SGVPDKFSGS GSGTDFTLKISRVEAEDLGV YFCFQGSHVP PTFGGGTKLE IK 152 15C6-A7 HVR-H1 DYYMK 15315C6-A7 HVR-H2 DLDPYTGGAN YNQKFKG 154 15C6-A7 HVR-H3 SRGYGDSFAY 15515C6-A7 HVR-L1 RSSQNIVHSN GNTYLE 156 15C6-A7 HVR-L2 KVSNRFS 15715C6-A7 HVR-L3 FQGSHVPPT 160 19F8-B1 heavyEVQLQQSGPE LVKPGASVKM SCKASGYTFT DYYMKWVKQS chain variableHGKSLEWIGD LNPNNGGTLY NQKFKGQATL TVDKSSSTAY region (VH)MQFNSLTSED SAVYYCARSA GYGDSFAYWG QGTLVTVSA 161 19F8-B1 light chainDVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW variable region (VL)YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKISRVEAEDLGI YFCFQGSHVP PTFGGGTKLE IK 162 19F8-B1 HVR-H1 DYYMK 16319F8-B1 HVR-H2 DLNPNNGGTL YNQKFKG 164 19F8-B1 HVR-H3 SAGYGDSFAY 16519F8-B1 HVR-L1 RSSQNIVHSN GNTYLE 166 19F8-B1 HVR-L2 KVSNRFS 16719F8-B1 HVR-L3 FQGSHVPPT 170 24A11-D5 heavyEVQLQQSGPE LVKPGASVKM SCKASGYTFT DYYMKWVKQS chain variableHGKSLEWIGD LNPKNGGIIY NQKFKGQATL TVDKSSSTAY region (VH)MQLNSLTSED SAVFYCARSG GYGDSFAYWG QGTLVTVSA 171 24A11-D5 lightDVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW chain variableYLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDLGI YFCFQGSHVP PTFGGGTKLE IK 172 24A11-D5 HVR-H1 DYYMK 17324A11-D5 HVR-H2 DLNPKNGGII YNQKFKG 174 24A11-D5 HVR-H3 SGGYGDSFAY 17524A11-D5 HVR-L1 RSSQNIVHSN GNTYLE 176 24A11-D5 HVR-L2 KVSNRFS 17724A11-D5 HVR-L3 FQGSHVPPT 180 126F11-G11 heavyEVQLQQSGAE LVRPGASVKL SCTASGFNIK DDYMHWVKQR chain variablePEQGLEWIGW IDPENGDTEY ASKFQGKATI TTDTSSNTAY region (VH)LQLSSLTSED TAVYYCLDFA YGYWGQGTTL TVSS 181 126F11-G11 lightDVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW chain variableYLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDLGV YYCFQGSHVP PAFGGGTKLE IK 182 126F11-G11 HVR- DDYMH H1 183126F11-G11 HVR- WIDPENGDTE YASKFQG H2 184 126F11-G11 HVR- FAYGY H3 185126F11-G11 HVR- RSSQSIVHSN GNTYLE L1 186 126F11-G11 HVR- KVSNRFS L2 187126F11-G11 HVR- FQGSHVPPA L3 190 89F4-A1 heavyEVQLVESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA chain variablePGKGLEWVAR IRSKSNNYAA YFADSVKDRF TISRDDSQTM region (VH)LYLQMNNLKS EDTAMYYCVS GGNYVPFAYW GQGTLVTVSA 191 89F4-A1 light chainNIMMTQSPSS LAVSAGEKVT MSCKSSQSVF YSSEQRNYLA variable region (VL)WYQQKPGQSP KLLISWASTR ESGVPDRFTG SGSGTDFTLTISSVQGEDLA VYYCHQYLSS FTFGSGTKLE IK 192 89F4-A1 HVR-H1 TYAMN 19389F4-A1 HVR-H2 RIRSKSNNYA AYFADSVKD 194 89F4-A1 HVR-H3 GGNYVPFAY 19589F4-A1 HVR-L1 KSSQSVFYSS EQRNYLA 196 89F4-A1 HVR-L2 WASTRES 19789F4-A1 HVR-L3 HQYLSSFT 200 93A8-D2 heavyEVQLQQSGPV LVKPGASVKM SCKASGYTFT DYYVNWVKQS chain variableHGKGLEWIGL INPNNGRTSY NQNFNDKATL TVDKSSSTAF region (VH)MDLNSLTSED SAVYYCTREG GTGYWGQGTT LSVSS 201 93A8-D2 light chainDVVMTQTPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW variable region (VL)LLQRPGQSPR RLIYLVSKLD SGVPDRFTGS GSGTDFTLKISRVAAEDLGV YYCWQGTHFP RTFGGGTKLE IK 202 93A8-D2 HVR-H1 DYYVN 20393A8-D2 HVR-H2 LINPNNGRTSYNQNFND 204 93A8-D2 HVR-H3 EGGTGY 20593A8-D2 HVR-L1 KSSQSLLDSDGKTYLN 206 93A8-D2 HVR-L2 LVSKLDS 20793A8-D2 HVR-L3 WQGTHFPRT 210 14F5-D9 heavyEVKLVESGGG LVQPGGSLRL SCATSGFTFS DFYMEWVRQS chain variablePGKRLEWIAA SKNKANDYTT EYNASVKDRF FVSRDTSQSI region (VH)LYLQMNALRA EDTAIYYCAR DALGTVFAYW GQGTLVTVSA 211 14F5-D9 light chainDVVMTQTPLS LPVSLGDQAS ISCRSSQSLV HSNGNTYLHW  variable region (VL)YLQKPGQSPK LLIYKVFNRF SGVPDRFSGS GSGTDFTLKISRVEAEDLGV YFCSQSTLVP LTFGAGTKLE LK 212 14F5-D9 HVR-H1 DFYME 21314F5-D9 HVR-H2 ASKNKANDYT TEYNASVKD 214 14F5-D9 HVR-H3 DALGTVFAY 21514F5-D9 HVR-L1 RSSQSLVHSN GNTYLH 216 14F5-D9 HVR-L2 KVFNRFS 21714F5-D9 HVR-L3 SQSTLVPLT 220 73H6-B8 heavyQVQLKESGPG LVAPSQSLSI TCTISGFSLT SYGVHWVRQP chain variablePGKGLEWLVV IWSDGSTTYN SALKSRLSIS KDNSKSQVFL region (VH)KMNSLQTDDT AMYYCARQGG FITTAYYAMD YWGQGTSVTV SS 221 73H6-B8 light chainDIVMSQSPSS LAVSAGEKVT MSCKSSQSLL NSRTRKNYLA variable region (VL)WYQQKPGQSP KLLIYWASTR ESGVPDRFTG SGSGTDFTLTISSVQAEDLA VYYCKQSYNL YTFGGGTKLE IK 222 73H6-B8 HVR-H1 SYGVH 22373H6-B8 HVR-H2 VIWSDGSTTY NSALKS 224 73H6-B8 HVR-H3 QGGFITTAYY ANDY 22573H6-B8 HVR-L1 KSSQSLLNSR TRKNYLA 226 73H6-B8 HVR-L2 WASTRES 22773H6-B8 HVR-L3 KQSYNLYT 230 22G7-C9 heavyQIQLVQSGPE LKKPGETVKI SCKASGYTFT DCSIHWVKQA chain variablePGEGLKWMGW INTETGEPSY ADDFKGRFAF SLETSASTAF region (VH)LQINNLKSED TASYFCGTAY YRYDGALDYW GQGTSVTVSS 231 22G7-C9 light chainDIVLTQSPAS LAVSLGQRAT ISCRASQSVS TSSYSYMHWF variable region (VL)QQKPGQPPKL LIKYASNLES GVPARFSGSG SGTDFTLNIHPVEEEDTATY YCQHSWELPW TFGGGTKLEI K 232 22G7-C9 HVR-H1 DCSIH 23322G7-C9 HVR-H2 WINTETGEPS YADDFKG 234 22G7-C9 HVR-H3 AYYRYDGALD Y 23522G7-C9 HVR-L1 RASQSVSTSS YSYMH 236 22G7-C9 HVR-L2 YASNLES 23722G7-C9 HVR-L3 QHSWELPWT 240 7A11-C12 heavyQIQLVQSGPD LKKPGETVKI SCKASGYTFT NYGMNWVKQA chain variablePGKGLKWMGW INTNTGEPTY AEEFKGRFAF SLETSASTAY region (VH)LQIDNLKNED TATYFCARGT VSFPYWGQGT LVTVSA 241 7A11-C12 lightDVVMSQTPLS LPVSLGDHAS ISCRSSQNLV HSDGNTYLHW chain variableYLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDLGV YFCSQSTHVI FTFGSGTKLE IK 242 7A11-C12 HVR-H1 NYGMN 2437A11-C12 HVR-H2 WINTNTGEPT YAEEFKG 244 7A11-C12 HVR-H3 GTVSFPY 2457A11-C12 HVR-L1 RSSQNLVHSD GNTYLH 246 7A11-C12 HVR-L2 KVSNRFS 2477A11-C12 HVR-L3 SQSTHVIFT 250 12A10-E8 heavyQIQLVQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQA chain variablePGKGLKWMGW INMYTGEPTY GDDFKGRFVF SLETSVSTVY region (VH)LQINNLKKED TATFFCARGG RPDYWGQGTS VTVSS 251 12A10-E8 lightDVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW chain variableYLQKPGQSPK LLIYKVFNRF SGVPDRFSGS GSGTDFTLKI region (VL)NRVEAEDLGV YYCLQGSHVP YTFGGGTKLE IK 252 12A10-E8 HVR-H1 NYGMN 25312A10-E8 HVR-H2 WINMYTGEPT YGDDFKG 254 12A10-E8 HVR-H3 GGRPDY 25512A10-E8 HVR-L1 RSSQSIVHSN GNTYLE 256 12A10-E8 HVR-L2 KVFNRFS 25712A10-E8 HVR-L3 LQGSHVPYT 260 55E7-F11 heavyEVKLEESGGG LVQPGGSMKL SCVASGFTFS NYWMNWVRQS chain variablePEKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKSS region (VH)VYLQMNNLRA EDTGIYYCAG YFYGGYFDVW GTGTTVTVSS 261 55E7-F11 lightELVLTQSPTT MAASPGKKIT ITCSASSSIS SNYLHWYQQK chain variablePGFSPKLLIY RTSNLASGVP ARFSGSGSGT SYSLTIGTME region (VL)AEDVATYYCQ QGSSLPFTFG SGTKLEIK 262 55E7-F11 HVR-H1 NYWMN 26355E7-F11 HVR-H2 QIRLKSDNYA THYAESVKG 264 55E7-F11 HVR-H3 YFYGGYFDV 26555E7-F11 HVR-L1 SASSSISSNY LH 266 55E7-F11 HVR-L2 RTSNLAS 26755E7-F11 HVR-L3 QQGSSLPFT 270 52F6-F11 heavyQVQLQQSGTE LAKPGASVKL SCKASGYTFT HYWMHWIKQR chain variablePGQGLEWIGY IYPTNDYTKY NQNFRDKATL TADESSNSAY region (VH)MQLNSLTYED SAVYYCARAG NRVFDFWGQG TTLTVSS 271 52F6-F11 lightQAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNFANWVQE chain variableKPDHLFTGLI GGTNNRAPGV PARFSGSLIG DKAALTITGA region (VL)QTEDEAIYFC ALWYSNLWVF GGGTKLTVL 272 52F6-F11 HVR-H1 HYWMH 27352F6-F11 HVR-H2 YIYPTNDYTK YNQNFRD 274 52F6-F11 HVR-H3 AGNRVFDF 27552F6-F11 HVR-L1 RSSTGAVTTS NFAN 276 52F6-F11 HVR-L2 GTNNRAP 27752F6-F11 HVR-L3 ALWYSNLWV 280 Hu37D3-H9.v1EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variablePGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH)LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 281 Hu37D3-H9.v1EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYFEW light chain variableYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL)SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IK 282 Hu37D3-H9.v1 SYGMS HVR-H1 283Hu37D3-H9.v1 TINSGGTYTYYPDSVKG HVR-H2 284 Hu37D3-H9.v1 SYSGAMDY HVR-H3285 Hu37D3-H9.v1 RSSQSIVHSNGNTYFE HVR-L1 286 Hu37D3-H9.v1 KVSNRFS HVR-L2287 Hu37D3-H9.v1 FQGSLVPWT HVR-L3 288 Hu37D3-H9.v1EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG1 heavy chainPGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLYLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK 289 Hu37D3-H9.v1EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYFEW IgG1 light chainYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTISSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 290Hu37D3-H9.v5 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQAheavy chain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLYregion (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 291 Hu37D3-H9.v5EDVLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYFEW light chain variableYLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDVGV YYCFQGSLVP WTFGQGTKVE IK 292 Hu37D3-H9.v5 SYGMS HVR-H1 293Hu37D3-H9.v5 TINSGGTYTYYPDSVKG HVR-H2 294 Hu37D3-H9.v5 SYSGAMDY HVR-H3295 Hu37D3-H9.v5 RSSQSIVHSNGNTYFE HVR-L1 296 Hu37D3-H9.v5 KVSNRFS HVR-L2297 Hu37D3-H9.v5 FQGSLVPWT HVR-L3 300 Hu94B2.v105 heavyEVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA chain variablePGQGLEWIGL ISPYNGVTSY NQKFKGRATL TVDKSTSTAY region (VH)LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 301 Hu94B2.v105 lightDIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW chain variableLLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 302 Hu94B2.v105 GYTMN HVR-H1 303Hu94B2.v105 LISPYNGVTSYNQKFKG HVR-H2 304 Hu94B2.v105 QGAY HVR-H3 305Hu94B2.v105 KSSQSLLDSDGKTYLN HVR-L1 306 Hu94B2.v105 LVSKLDS HVR-L2 307Hu94B2.v105 WQGTHFPWT HVR-L3 310 hu125B11.v17EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA heavy chainPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNT variable regionLYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS (VH) 311 hu125B11.v17DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP light chainGKSPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP variable regionEDFATYFCQQ FRTYPYTFGQ GTKVEIK (VL) 312 hu125B11.v17 NYWMN HVR-H1 313hu125B11.v17 QIRLKSDNYATHYAESVKG HVR-H2 314 hu125B11.v17 GTTY HVR-H3 315hu125B11.v17 KASQNVGTAVA HVR-L1 316 hu125B11.v17 SASIRYT HVR-L2 317hu125B11.v17 QQFRTYPYT HVR-L3 320 hu125B11.v26EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA heavy chainPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDNSKNT variable regionLYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS (VH) 321 hu125B11.v26DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP light chainGKAPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP variable regionEDFATYFCQQ FRTYPYTFGQ GTKVEIK (VL) 322 hu125B11.v26 NYWMN HVR-H1 323hu125B11.v26 QIRLKSDNYATHYAESVKG HVR-H2 324 hu125B11.v26 GTTY HVR-H3 325hu125B11.v26 KASQNVGTAVA HVR-L1 326 hu125B11.v26 SASIRYT HVR-L2 327hu125B11.v26 QQFRTYPYT HVR-L3 330 hu125B11.v28EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA heavy chainPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDNSKNT variable regionLYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS (VH) 331 hu125B11.v28DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP light chainGKAPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP variable regionEDFATYYCQQ FRTYPYTFGQ GTKVEIK (VL) 332 hu125B11.v28 NYWMN HVR-H1 333hu125B11.v28 QIRLKSDNYATHYAESVKG HVR-H2 334 hu125B11.v28 GTTY HVR-H3 335hu125B11.v28 KASQNVGTAVA HVR-L1 336 hu125B11.v28 SASIRYT HVR-L2 337hu125B11.v28 QQFRTYPYT HVR-L3 340 Hu37D3-H9.v28.A4EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variablePGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH)LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 341 Hu37D3-H9.v28.A4DDVLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYLEW light chain variableYLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDVGV YYCFQGSLVP WTFGQGTKVE IK 342 Hu37D3-H9.v28.A4 SYGMS HVR-H1343 Hu37D3-H9.v28.A4 TINSGGTYTYYPDSVKG HVR-H2 344 Hu37D3-H9.v28.A4SYSGAMDY HVR-H3 345 Hu37D3-H9.v28.A4 RSSQSIVHSNGNTYLE HVR-L1 346Hu37D3-H9.v28.A4 KVSNRFS HVR-L2 347 Hu37D3-H9.v28.A4 FQGSLVPWT HVR-L3348 Hu37D3-H9.v28.A4 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQAIgG4-8228P.YTE PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY heavy chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 602 Hu37D3-H9.v28.A4EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-8228P.YTEPGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY des-K heavy chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG 349 Hu37D3-H9.v28.A4DDVLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYLEW IgG4-8228P.YTEYLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI light chainSRVEAEDVGV YYCFQGSLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 442hu125B11-H3.LC1 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKPGKSPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQPEDFATYFCQQ FRTYPYTFGQ GTKVEIK 443 hu125B11-H3.LC2DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKPGKAPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQPEDFATYFCQQ FRTYPYTFGQ GTKVEIK 444 hu125B11-H3.LC3DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKPGKSPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ FRTYPYTFGQ GTKVEIK 445 hu125B11-H3.LC4DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKPGKAPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ FRTYPYTFGQ GTKVEIK 446 hu125B11-H3.HC1EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQAPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNTVYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 447 hu125B11-H3.HC2EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQAPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDNSKNTVYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 448 hu125B11-H3.HC3EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQAPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNTLYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 449 hu125B11-H3.HC4EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQAPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDNSKNTLYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 450 hu125B11-H3.HC5EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYYMNWVRQAPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNTVYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 451 hu125B11-H3.HC6EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYFMNWVRQAPGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNTVYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 452 Hu94B2.HC1EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQAPGQGLEWIGL ISPYNGVTSY NQKFKGRATL TVDKSTSTAYLELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 453 Hu94B2.HC2EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQAPGQGLEWIGL ISPYNGVTSY NQKFKGRVTL TVDKSTSTAYLELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 454 Hu94B2.HC3EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQAPGQGLEWIGL ISPYNGVTSY NQKFKGRATI TVDKSTSTAYLELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 455 Hu94B2.HC4EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQAPGQGLEWIGL ISPYNGVTSY NQKFKGRATL TRDKSTSTAYLELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 456 Hu94B2.HC5EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQAPGQGLEWIGL ISPYNGVTSY NQKFKGRATL TVDTSTSTAYLELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 457 Hu94B2.HC6EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQAPGQGLEWIGL ISPYNGVTSY NQKFKGRVTI TVDKSTSTAYLELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 458 Hu94B2.HC7EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQAPGQGLEWIGL ISPYNGVTSY NQKFKGRVTI TRDKSTSTAYLELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 459 Hu94B2.HC8EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQAPGQGLEWIGL ISPYNGVTSY NQKFKGRVTI TVDTSTSTAYLELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 460 Hu94B2.LC9DVVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNWLLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 461 Hu94B2.LC10DVVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNWLLQKPGQSPQ LLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 462 Hu94B2.LC11DVVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNWYLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 463 Hu94B2.LC12DVVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNWYLQKPGQSPQ LLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 464 Hu94B2.LC13DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNWLLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 465 Hu94B2.LC14DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNWLLQKPGQSPQ LLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 466 Hu94B2.LC15DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNWYLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 467 Hu94B2.LC16DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNWYLQKPGQSPQ LLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 468 Hu37D3-H9.v5.1 RSSQSIVHSNANTYFEHVR-L1 469 Hu37D3-H9.v5.2 RSSQSIVHSSGNTYFE HVR-L1 470 Hu37D3-H9.v5.3RSSQSIVHSDGNTYFE HVR-L1 471 Hu37D3-H9.v5.4 RSSQSIVHSQGNTYFE HVR-L1 472Hu37D3-H9.v5.5 RSSQSIVHSEGNTYFE HVR-L1 473 Hu37D3-H9.v5.6RSSQSIVHSAGNTYFE HVR-L1 474 Hu37D3-H9.v5.7 RSSQSIVHSNGDTYFE HVR-L1 475Hu37D3-H9.v5.8 RSSQSIVHSNGQTYFE HVR-L1 476 Hu37D3-H9.v5.9RSSQSIVHSNGETYFE HVR-L1 477 Hu37D3-H9.v5.10 RSSQSIVHSNGATYFE HVR-L1 478Hu37D3-H9.v5.11 RSSQSIVHSNGSTYFE HVR-L1 479 Hu37D3.v28 RSSQSIVHSNGNTYFEHVR-L1 480 Hu37D3.v28.A2 RSSQSIVHSNGNTYFE HVR-L1 481 Hu37D3.v28.A4RSSQSIVHSNGNTYLE HVR-L1 482 Hu37D3.v28.A6 RSSQSIVHSNGNTYLE HVR-L1 483Hu37D3.v28.A8 RSSQSIVHSNGNTYFE HVR-L1 484 Hu37D3.v28.A10RSSQSIVHSNGNTYFE HVR-L1 485 Hu37D3.v28.A12 RSSQSIVHSNGNTYLE HVR-L1 486Hu37D3.v28.A14 RSSQSIVHSNGNTYLE HVR-L1 487 Hu37D3.v28.A16RSSQSIVHSNGNTYFE HVR-L1 488 Hu37D3.v28.A18 RSSQSIVHSNGNTYFE HVR-L1 489Hu37D3.v28.A20 RSSQSIVHSNGNTYLE HVR-L1 490 Hu37D3.v28.A22RSSQSIVHSNGNTYLE HVR-L1 491 Hu37D3.v28.A24 RSSQSIVHSNGNTYFE HVR-L1 492Hu37D3.v28.A26 RSSQSIVHSNGNTYFE HVR-L1 493 Hu37D3.v28.A28RSSQSIVHSNGNTYLE HVR-L1 494 Hu37D3.v28.A30 RSSQSIVHSNGNTYLE HVR-L1 495Hu37D3.v28.B1 RSSQSIVHSIGNTFFE HVR-L1 496 Hu37D3.v28.B2 RSSQSIVHSMGNTFFEHVR-L1 497 Hu37D3.v28.B3 RSSQSIVHSQGNTWFE HVR-L1 498 Hu37D3.v28.B4RSSQSIVHSQGNTHFE HVR-L1 499 Hu37D3.v28.B6 RSSQSIVHSDGNTRFE HVR-L1 500Hu37D3.v28.B7 RSSQSIVHSDGNIKFE HVR-L1 501 Hu37D3.v28.B8 RSSQSIVHSEGNTRFEHVR-L1 502 Hu37D3.v28.C1 RSSQSIVHSNNNTYFE HVR-L1 503 Hu37D3.v28.C2RSSQSIVHSNDNTYFE HVR-L1 504 Hu37D3.v28.D1 RSSQSIVHANGNTYFE HVR-L1 505Hu37D3.v28.E1 RSSQSIVNSNGNTYFE HVR-L1 506 Hu37D3.v28.E2 RSSQSIVQSNGNTYFEHVR-L1 507 Hu37D3.v28.E3 RSSQSIVDSDGNTYFE HVR-L1 508 Hu37D3.v28.F1RSSQSIVHSNTNTYFE HVR-L1 509 Hu37D3.v28.F2 RSSQSIVHTNGNTYFE HVR-L1 510Hu37D3.v28.F3 RSSQSIVHTNANTYFE HVR-L1 511 Hu37D3.v28.51 RSSQSIVHSHGNTYFEHVR-L1 512 Hu37D3.v28.52 RSSQSIVHSKGNTYFE HVR-L1 513 Hu37D3.v28.53RSSQSIVHSRGNTYFE HVR-L1 514 Hu37D3.v28.54 RSSQSIVHSLGNTYFE HVR-L1 515Hu37D3.v28.55 RSSQSIVHSNQNTYFE HVR-L1 516 Hu37D3.v28.56 RSSQSIVHSNYNTYFEHVR-L1 517 Hu37D3.v28.57 RSSQSIVHSNFNTYFE HVR-L1 518 Hu37D3.v29.1RSSQSIVHSNGDTYFE HVR-L1 519 Hu37D3.v29.2 RSSQSIVHSNGQTYFE HVR-L1 520Hu37D3.v29.3 RSSQSIVHSNGETYFE HVR-L1 521 Hu37D3.v29.4 RSSQSIVHSNGATYFEHVR-L1 522 Hu37D3.v29.5 RSSQSIVHSNGHTYFE HVR-L1 523 Hu37D3.v29.6RSSQSIVHSNGKTYFE HVR-L1 524 Hu37D3.v29.7 RSSQSIVHSNGLTYFE HVR-L1 525Hu37D3.v29.8 RSSQSIVHSNADTYFE HVR-L1 526 Hu37D3.v29.9 RSSQSIVHSNAQTYFEHVR-L1 527 Hu37D3.v29.10 RSSQSIVHSNAETYFE HVR-L1 528 Hu37D3.v29.11RSSQSIVHSNAATYFE HVR-L1 529 Hu37D3.v29.12 RSSQSIVHSNAHTYFE HVR-L1 530Hu37D3.v29.13 RSSQSIVHSNAKTYFE HVR-L1 531 Hu37D3.v29.14 RSSQSIVHSNALTYFEHVR-L1 532 Hu37D3-H9.v30.1 RSSQSIVHSGGNTYFE HVR-L1 533 Hu37D3-H9.v30.2RSSQSIVHSTGNTYFE HVR-L1 534 Hu37D3-H9.v30.3 RSSQSIVHSVGNTYFE HVR-L1 535Hu37D3-H9.v30.4 RSSQSIVHSLGNTYFE HVR-L1 536 Hu37D3-H9.v30.5RSSQSIVHSIGNTYFE HVR-L1 537 Hu37D3-H9.v30.6 RSSQSIVHSPGNTYFE HVR-L1 538Hu37D3-H9.v30.7 RSSQSIVHSFGNTYFE HVR-L1 539 Hu37D3-H9.v30.8RSSQSIVHSYGNTYFE HVR-L1 540 Hu37D3-H9.v30.9 RSSQSIVHSHGNTYFE HVR-L1 541Hu37D3-H9.v30.10 RSSQSIVHSKGNTYFE HVR-L1 542 Hu37D3-H9.v30.11RSSQSIVHSRGNTYFE HVR-L1 543 Hu37D3-H9.v31.1 RSSQSIVHSNAGTYFE HVR-L1 544Hu37D3-H9.v31.2 RSSQSIVHSNAVTYFE HVR-L1 545 Hu37D3-H9.v31.3RSSQSIVHSNAITYFE HVR-L1 546 Hu37D3-H9.v31.4 RSSQSIVHSNAPTYFE HVR-L1 547Hu37D3-H9.v31.5 RSSQSIVHSNAFTYFE HVR-L1 548 Hu37D3-H9.v31.6RSSQSIVHSNAYTYFE HVR-L1 549 Hu37D3-H9.v31.7 RSSQSIVHSNARTYFE HVR-L1 550Hu37D3-H9.v31.8 RSSQSIVHSNANVYFE HVR-L1 551 Hu37D3-H9.v31.9RSSQSIVHSNANIYFE HVR-L1 552 Hu37D3-H9.v31.10 RSSQSIVHSNANPYFE HVR-L1 553Hu37D3-H9.v31.11 RSSQSIVHSNANFYFE HVR-L1 554 Hu37D3-H9.v31.12RSSQSIVHSNANYYFE HVR-L1 555 Hu37D3-H9.v31.13 RSSQSIVHSNANNYFE HVR-L1 556Hu37D3-H9.v31.14 RSSQSIVHSNANRYFE HVR-L1 557 Human Tau 7-24EFEVMEDHAGTYGLGDRK peptide 558 Human Tau 7-20 EFEVMEDHAGTYGL peptide 560Hu37D3.v39 heavy EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQAchain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH)LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 561 Hu37D3.v39 lightEDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYLEW chain variableYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL)SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IK 562 Hu37D3.v39 HVR- SYGMS H1 563Hu37D3.v39 HVR- TINSGGTYTYYPDSVKG H2 564 Hu37D3.v39 HVR- SYSGAMDY H3 565Hu37D3 .v39 FIVR- RSSQSIVHSNGNTYLE L1 566 Hu37D3.v39 HVR- KVSNRFS L2 567Hu37D3.v39 HVR- FQGSLVPWT L3 568 Hu37D3.v39 IgG4-EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA S228P.YTE heavyPGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 569 Hu37D3.v39 IgG4-EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYLEW S228P.YTE lightYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI chainSSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 570Hu37D3.v40 heavy EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQAchain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH)LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 571 Hu37D3.v40 lightEDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNTNTYFEW chain variableYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL)SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IK 572 Hu37D3.v40 HVR- SYGMS H1 573Hu37D3.v40 HVR- TINSGGTYTYYPDSVKG H2 574 Hu37D3.v40 HVR- SYSGAMDY H3 575Hu37D3.v40 HVR- RSSQSIVHSNTNTYFE L1 576 Hu37D3.v40 HVR- KVSNRFS L2 577Hu37D3.v40 HVR- FQGSLVPWT L3 578 Hu37D3.v40 IgG4-EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA S228P.YTE heavyPGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 579 Hu37D3.v40 IgG4-EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNTNTYFEW S228P.YTE lightYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI chainSSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 580Hu37D3.v41 heavy EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQAchain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH)LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 581 Hu37D3.v41 lightEDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGQTYFEW chain variableYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL)SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IK 582 Hu37D3.v41 HVR- SYGMS H1 583Hu37D3.v41 HVR- TINSGGTYTYYPDSVKG H2 584 Hu37D3.v41 HVR- SYSGAMDY H3 585Hu37D3.v41 HVR- RSSQSIVHSNGQTYFE L1 586 Hu37D3.v41 HVR- KVSNRFS L2 587Flu37D3.v41FIVR- FQGSLVPWT L3 588 Hu37D3.v41 IgG4-EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA S228P.YTE heavyPGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 589 Hu37D3.v41 IgG4-EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGQTYFEW S228P.YTE lightYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI chainSSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 590Hu37D3-H9.v1 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQAIgG4-S228P heavy PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 591 Hu37D3-H9.v1EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYFEW IgG4 light chainYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTISSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 592MAPT(10-24) VMEDHAGTYGLGDRK 593 MAPT(2-24) AEPRQEFEVMEDHAGTYGLGDRK 594MAPT(2-34) AEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD 595 MAPT(10-44)VMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK 596 MAPT(2-24)Y18AAEPRQEFEVMEDHAGTAGLGDRK 597 MAPT(2-24)L20A AEPRQEFEVMEDHAGTYGAGDRK 598hu113F5-F7.LC1 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKPGKSPKLLIYS ASRRFSGVPS RFSGSGSGTD FTLTISSLQPEDFATYFCQQ FSTYPYTFGQ GTKVEIK 599 hu113F5-F7.LC2DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKPGKAPKLLIYS ASRRFSGVPS RFSGSGSGTD FTLTISSLQPEDFATYFCQQ FSTYPYTFGQ GTKVEIK 600 hu113F5-F7.LC3DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKPGKSPKLLIYS ASRRFSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ FSTYPYTFGQ GTKVEIK 601 hu113F5-F7.LC4DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKPGKAPKLLIYS ASRRFSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ FSTYPYTFGQ GTKVEIK 603 Hu37D3-H9.v76EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variablePGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY region (VH)LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 604 Hu37D3-H9.v76DDLLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYLEW light chain variableYLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDVGV YYCFQGTLVP WTFGQGTKVE IK 605 Hu37D3-H9.v76 SYGMS HVR-H1Hu37D3-H9.v83 HVR-H1 Hu37D3-H9.v93 HVR-H1 606 Hu37D3-H9.v76TINSGGTRTYYPDSVKG HVR-H2 Hu37D3-H9.v83 HVR-H2 Hu37D3-H9.v93 HVR-H2 607Hu37D3-H9.v76 SYSGAMDY HVR-H3 Hu37D3-H9.v83 HVR-H3 Hu37D3-H9.v93 HVR-H3608 Hu37D3-H9.v76 RSSQSIVHSNGNTYLE HVR-L1 Hu37D3-H9.v83 HVR-L1Hu37D3-H9.v93 HVR-L1 609 Hu37D3-H9.v76 KVSNRFS HVR-L2 Hu37D3-H9.v83HVR-L2 Hu37D3-H9.v93 HVR-L2 610 Hu37D3-H9.v76 FQGTLVPWT HVR-L3Hu37D3-H9.v83 HVR-L3 Hu37D3-H9.v93 HVR-L3 611 Hu37D3-H9.v76EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTEPGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY heavy chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 612 Hu37D3-H9.v76EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTEPGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY des-K heavy chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG 613 Hu37D3-H9.v76DDLLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYLEW IgG4-S228P.YTEYLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI light chainSRVEAEDVGV YYCFQGTLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 614Hu37D3-H9.v83 EVQLLESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQAheavy chain variable PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLYregion (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 615 Hu37D3-H9.v83DDLLTQSPLS LPVTLGQPAS ISCRSSQSIV HSNGNTYLEW light chain variableYQQRPGQSPR LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL)SRVEAEDVGV YYCFQGTLVP WTFGQGTKVE IK 616 Hu37D3-H9.v83EVQLLESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTEPGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY heavy chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 617 Hu37D3-H9.v83EVQLLESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTEPGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY des-K heavy chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG 618 Hu37D3-H9.v83DDLLTQSPLS LPVTLGQPAS ISCRSSQSIV HSNGNTYLEW IgG4-S228P.YTEYQQRPGQSPR LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI light chainSRVEAEDVGV YYCFQGTLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 619Hu37D3-H9.v93 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQAheavy chain variable PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLYregion (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 620 Hu37D3-H9.v93EDLLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYLEW light chain variableYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL)SSLQPEDFAT YYCFQGTLVP WTFGQGTKVE IK 621 Hu37D3-H9.v93EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTEPGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY heavy chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 622 Hu37D3-H9.v93EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTEPGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY des-K heavy chainLQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNSGALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLFPPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG Hu37D3-H9.v93EDLLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYLEW IgG4-S228P.YTEYQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI 623 light chainSSLQPEDFAT YYCFQGTLVP WTFGQGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 624residues 2 to 24 of AEPRQEFEVMEDHAGTYGLGDRK human Tau

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. A method of treating atauopathy comprising administering to an individual with a tauopathy anisolated antibody that binds to human Tau, wherein the antibodycomprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 605;HVR-H2 comprising the amino acid sequence of SEQ ID NO: 606; HVR-H3comprising the amino acid sequence of SEQ ID NO: 607; HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 608; HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 609; and HVR-L3 comprising the amino acidsequence of SEQ ID NO: 610, and wherein the antibody is humanized. 5.The method of claim 4, wherein the antibody binds to monomeric Tau,oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau.
 6. Themethod of claim 4, wherein the antibody binds an epitope within aminoacids 2 to 24 of mature human Tau.
 7. The method of claim 4, wherein theantibody is a monoclonal antibody.
 8. (canceled)
 9. The method of claim4, wherein the antibody any one of the preceding claims, which is anantibody fragment that binds human Tau.
 10. The method of claim 4,wherein the human Tau comprises the sequence of SEQ ID NO:
 2. 11. Themethod of claim 4, wherein the antibody comprises: a) a heavy chainvariable region (VH) comprising a sequence that is at least 95%identical to SEQ ID NO: 603; b) a light chain variable region (VL)comprising a sequence that is at least 95% identical to SEQ ID NO: 604;c) a VH as in (a) and a VL as in (b); d) a heavy chain variable region(VH) comprising a sequence that is at least 95% identical to SEQ ID NO:614; e) a light chain variable region (VL) comprising a sequence that isat least 95% identical to SEQ ID NO: 615; f) a VH as in (d) and a VL asin (e); g) a heavy chain variable region (VH) comprising a sequence thatis at least 95% identical to SEQ ID NO: 619; h) a light chain variableregion (VL) comprising a sequence that is at least 95% identical to SEQID NO: 620; i) a VH as in (g) and a VL as in (h).
 12. The method ofclaim 4, wherein the antibody comprises: a) a heavy chain variableregion (VH) comprising SEQ ID NO: 603; b) a light chain variable region(VL) comprising SEQ ID NO: 604; c) a VH as in (a) and a VL as in (b); d)a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:614; e) a light chain variable region (VL) comprising the sequence ofSEQ ID NO: 615; f) a VH as in (d) and a VL as in (e); g) a heavy chainvariable region (VH) comprising the sequence of SEQ ID NO: 619; h) alight chain variable region (VL) comprising the sequence of SEQ ID NO:620; i) a VH as in (g) and a VL as in (h).
 13. The method of claim 4,wherein the antibody comprises a heavy chain variable region comprisinga sequence selected from SEQ ID NOs: 603, 614, and 619; and a lightchain variable region comprising a sequence selected from SEQ ID NOs:604, 615, and
 620. 14. The method of claim 4, wherein the antibodycomprises a heavy chain variable region comprising a sequence selectedfrom SEQ ID NOs: 340, 603, 614, and 619; and a light chain variableregion comprising a sequence selected from SEQ ID NOs: 604, 615, and620.
 15. The method of claim 4, wherein the antibody comprises a heavychain variable region comprising a sequence selected from SEQ ID NOs:603, 614, and 619; and a light chain variable region comprising asequence selected from SEQ ID NOs: 341, 604, 615, and
 620. 16. Themethod of claim 4, wherein the antibody comprises (a) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 340 anda light chain variable region comprising a sequence selected from SEQ IDNOs: 604, 615, and 620; (b) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 603 and a light chain variable regioncomprising a sequence selected from SEQ ID NOs: 341, 604, 615, and 620;(c) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 614 and a light chain variable region comprising a sequenceselected from SEQ ID NOs: 341, 604, 615, and 620; (d) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 619 anda light chain variable region comprising a sequence selected from SEQ IDNOs: 341, 604, 615, and 620; (e) a heavy chain variable regioncomprising a sequence selected from SEQ ID NOs: 603, 614, and 619 and alight chain variable region comprising the amino acid sequence of SEQ IDNO: 341; (f) a heavy chain variable region comprising a sequenceselected from SEQ ID NOs: 340, 603, 614, and 619 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 604;(g) a heavy chain variable region comprising a sequence selected fromSEQ ID NOs: 340, 603, 614, and 619 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 615; and (h) a heavychain variable region comprising a sequence selected from SEQ ID NOs:340, 603, 614, and 619 and a light chain variable region comprising theamino acid sequence of SEQ ID NO:
 620. 17. The method of claim 4,wherein the antibody comprises (a) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 603 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 604;(b) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 614 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 615; or (c) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 619 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 620.18. A method of treating a tauopathy comprising administering to anindividual with a tauopathy an antibody that binds to human Tau, whereinthe antibody comprises (a) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 603 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 604; (b) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 614 anda light chain variable region comprising the amino acid sequence of SEQID NO: 615; or (c) a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 619 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 620. 19. The method ofclaim 4, wherein the antibody comprises: a) a heavy chain comprising anamino acid sequence that is at least 95%, at least 97%, or at least 99%identical to the sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and alight chain comprising an amino acid sequence that is at least 95%, atleast 97%, or at least 99% identical to the sequence of SEQ ID NO: 613;or b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 611or SEQ ID NO: 612 and a light chain comprising the amino acid sequenceof SEQ ID NO: 613; or c) a heavy chain comprising an amino acid sequencethat is at least 95%, at least 97%, or at least 99% identical to thesequence of SEQ ID NO: 616 or SEQ ID NO: 617 and a light chaincomprising an amino acid sequence that is at least 95%, at least 97%, orat least 99% identical to the sequence of SEQ ID NO: 618; or d) a heavychain comprising the amino acid sequence of SEQ ID NO: 616 or SEQ ID NO:617 and a light chain comprising the amino acid sequence of SEQ ID NO:618; or e) a heavy chain comprising an amino acid sequence that is atleast 95%, at least 97%, or at least 99% identical to the sequence ofSEQ ID NO: 621 or SEQ ID NO: 622 and a light chain comprising an aminoacid sequence that is at least 95%, at least 97%, or at least 99%identical to the sequence of SEQ ID NO: 623; or f) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622and a light chain comprising the amino acid sequence of SEQ ID NO: 623.20. A method of treating a tauopathy comprising administering to anindividual with a tauopathy an antibody that binds to human Tau, whereinthe antibody comprises (a) a heavy chain comprising the amino acidsequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a light chaincomprising the amino acid sequence of SEQ ID NO: 613; (b) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 616 or SEQ ID NO: 617and a light chain comprising the amino acid sequence of SEQ ID NO: 618;or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO:621 or SEQ ID NO: 622 and a light chain comprising the amino acidsequence of SEQ ID NO:
 623. 21. A method of treating a tauopathycomprising administering to an individual with a tauopathy an antibodythat binds to human Tau, wherein the antibody comprises a heavy chainconsisting of the amino acid sequence of SEQ ID NO: 611 or SEQ ID NO:612 and a light chain consisting of the amino acid sequence of SEQ IDNO: 613; (b) a heavy chain consisting of the amino acid sequence of SEQID NO: 616 or SEQ ID NO: 617 and a light chain consisting of the aminoacid sequence of SEQ ID NO: 618; or (c) a heavy chain consisting of theamino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a lightchain consisting of the amino acid sequence of SEQ ID NO:
 623. 22. Themethod of claim 4, wherein the antibody is an IgG₁ or an IgG₄ antibody.23. The method of claim 22, wherein the antibody is an IgG₄ antibody.24. The method of claim 23, wherein the antibody comprises M252Y, S254T,and T256E mutations.
 25. The method of claim 24, wherein the antibodycomprises an S228P mutation.
 26. The method of claim 12, wherein theantibody is an antibody fragment that binds human Tau.
 27. The method ofclaim 4, wherein the antibody binds each of monomeric Tau,phosphorylated Tau, non-phosphorylated Tau, and oligomeric Tau with aK_(D) of less than 5 nM.
 28. The method of claim 4, wherein the antibodybinds to human monomeric Tau with a K_(D) of less than 1 nM.
 29. Themethod of claim 27, wherein K_(D) is determined by surface plasmonresonance at 37° C.
 30. The method of claim 4, wherein the antibodybinds cynomolgus monkey Tau. 31.-39. (canceled)
 40. The method of claim18, wherein the tauopathy is selected from Alzheimer's Disease,amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jacobdisease, Dementia pugilistica, Down Syndrome,Gerstmann-Straussler-Scheinker disease, inclusion-body myositis, prionprotein cerebral amyloid angiopathy, traumatic brain injury, amyotrophiclateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanianmotor neuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangleswith calcification, frontotemporal dementia, frontotemporal dementiawith parkinsonism linked to chromosome 17, Hallervorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy.
 41. The method of claim 18, wherein thetauopathy is Alzheimer's disease or progressive supranuclear palsy. 42.(canceled)
 43. (canceled)
 44. The method of claim 18, wherein the methodcomprises administering at least one additional therapy.
 45. The methodof claim 44, wherein the additional therapy is selected fromneurological drugs, corticosteroids, antibiotics, antiviral agents,anti-Tau antibodies, Tau inhibitors, anti-amyloid beta antibodies,beta-amyloid aggregation inhibitors, anti-BACE1 antibodies, and BACE1inhibitors.
 46. (canceled)
 47. (canceled)
 48. (canceled)
 49. The methodof claim 20, wherein the tauopathy is selected from Alzheimer's Disease,amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jacobdisease, Dementia pugilistica, Down Syndrome,Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis, prionprotein cerebral amyloid angiopathy, traumatic brain injury, amyotrophiclateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanianmotor neuron disease with neurofibrillary tangles, argyrophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangleswith calcification, frontotemporal dementia, frontotemporal dementiawith parkinsonism linked to chromosome 17, Hallervorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,Pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy, Subacute sclerosingpanencephalitis, Tangle only dementia, Postencephalitic Parkinsonism,and Myotonic dystrophy.
 50. The method of claim 20, wherein thetauopathy is Alzheimer's disease or progressive supranuclear palsy. 51.(canceled)
 52. (canceled)
 53. The method of claim 20, wherein the methodcomprises administering aat least one additional therapy.
 54. The methodof claim 53, wherein the additional therapy is selected fromneurological drugs, corticosteroids, antibiotics, antiviral agents,anti-Tau antibodies, anti-amyloid beta antibodies, anti-BACE1antibodies, and BACE1 inhibitors. 55.-64. (canceled)